CN100447151C

CN100447151C - Peptide boronic acids useful in making salts thereof

Info

Publication number: CN100447151C
Application number: CNB038213907A
Authority: CN
Inventors: 戴维·乔纳森·玛奇; 马克·多尔曼; 索菲·玛丽·库姆-马泽勒; 约翰·约瑟夫·戴德曼; 安东尼·詹姆斯·肯尼迪; 桑贾伊·库马尔·卡卡尔
Original assignee: Trigen Ltd
Current assignee: Trigen Ltd
Priority date: 2002-09-09
Filing date: 2003-09-09
Publication date: 2008-12-31
Anticipated expiration: 2023-09-09
Also published as: SI1400245T1; EP1396270B1; HK1063154A1; DE60317039D1; CN1684693A; SI1396270T1; DE60305113D1; PT1396270E; DE60317039T2; WO2004022071A1; EP1466916A1; IL167294A; DE60304956D1; CA2536010A1; EP1396270A1; CN1681517A; EP1466916B1; EP1695712A2; EP1466917A1; EP1561466A3

Abstract

Salts of a peptide boronic acid drug, for example of Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)2. The counter-ion to the boronate may be an alkali metal or derived from an organic nitrogen-containing compound. The salts are used for the manufacture of a medicament for treating thrombosis.

Description

Borate and the application in preparation treatment thrombosis medicine thereof

Technical field

The present invention relates to derive from the useful product of pharmacology of organic boronic.The invention still further relates to the application of each member in the aforementioned class product, relate to its preparation, its preparation, its synthetic intermediate and relate to other theme.

The invention further relates to the oral drug preparation that contains described product.

Background technology

Boronic acid compounds

In these years known boric acid (boronic acid) compound and derivative thereof for example ester have biologic activity, particularly as proteinase inhibitor or substrate.For example, Koehler etc., Biochemistry 10:2477,1971 have reported that 2-phenylethane boric acid suppresses the serine protease Quimotrase in the mmole level.Phillip etc., Proc.Nat.Acad.Sci.USA 68:478-480,1971 have reported the inhibition of aryl boric acid (phenyl-boron dihydroxide, nitro-phenyl-boron dihydroxide, m-aminophenyl ylboronic acid, a bromophenyl boric acid) to Quimotrase and subtilisin.Seufer-Wasserthal etc., Biorg.Med.Chem.2 (1): 35-48,1994 have described multiple boric acid, especially by Cl, Br, CH ₃, H ₂The research that the phenyl-boron dihydroxide that N, MeO etc. replace suppresses subtilisin Carisberg.

In the inhibitor or substrate of describing proteolytic enzyme, P1, P2, P3 etc. are meant the aminoterminal substrate or the inhibitor residue of the peptide bond of easy fracture, S1, S2, S3 etc. are meant according to Schechter, I.and Berger, A.On the Size of the Active Site in Proteases, Biochem.Biophys.Res.Comm., 27:157-162, the corresponding sublocus of 1967 described connection proteolytic enzyme.In zymoplasm, S1 binding site or " specificity pocket " are meant the crack in the described enzyme, simultaneously S2 and S3 are hydrophobic in conjunction with sublocus (also being called near-end and far-end hydrophobicity pocket), and for example respectively and (R) with Pro-and Phe reacts forcefully.

Pharmacology research to serpin is transferred to boric acid peptide (boropeptide) from simple aryl boric acid, promptly contains the peptide of the boric acid analogue of alpha-amino carboxylic acid.Described boric acid can be deutero-, forms a kind of ester usually.Shenvi (EP-A-145441 and US4499082) has disclosed effective inhibitor that the peptide that contains a kind of alpha-amino boronic acid with neutral side chain is an elastoser, and the patent publications of many boric acid inhibitor peptides about serine protease is arranged subsequently.Especially, reported the boric acid inhibitor of combining closely of following enzyme: elastoser (Ki, 0.25nM), Quimotrase (Ki, 0.25nM), (Ki of cathepsin G, 21nM), α-crack protein enzyme (Ki, 0.25nM), IV type two peptidyls (dipeptidyl) aminopeptidase (Ki, 16pM) and more nearest zymoplasm (Ac-D-Phe-Pro-boroArg-OH (DuP 714, initial Ki 1.2nM).

(WO 92/07869 and patent families comprise US 5648338) such as Claeson etc. (US 5574014 etc.) and Kakkar disclosed and had for example thrombin inhibitors of alkyl or alkoxyalkyl side chain of neutral C-terminal side chain.

The modification that WO 96/25427 has disclosed described compounds such as Kakkar is carried out at the peptidyl serpin, and wherein the natural peptide bond of P2-P1 is by another key displacement.The non-natural peptide bond for example can be-CO ₂-,-CH ₂O-,-NHCO-,-CHYCH ₂-,-CH=CH-,-CO (CH ₂) pCO-, wherein p be 1,2 or 3 ,-COCHY-,-CO ₂-CH ₂NH-,-CHY-NX-,-N (X) CH ₂-N (X) CO-,-CH=C (CN) CO-,-CH (OH)-NH-,-CH (CN)-NH-,-CH (OH)-CH ₂-or-NH-CHOH-, wherein X is H or amino protecting group, Y is H or halogen, especially F.Special non-natural peptide bond is-CO ₂-or-CH ₂O-.

Metternich (EP 471651 and US 5288707, back one patent is transferred to TrigenLimited) disclosed the variant of Phe-Pro-BoroArg boric acid peptide, wherein P3Phe is by a non-natural hydrophobic amino acid such as trimethyl silyl L-Ala, right-tertiary butyl-xenyl-silyloxy methylphenylalanine or right-hydroxymethyl phenylalanine displacement, and the P1 side chain can be neutral (alkoxyalkyl, alkyl-thio-alkyl or a trimethyl silyl alkyl).

Fevig J M et al Bioorg.Med.Chem.8:301-306 and Rupin A et alThromb.Haemost.78 (4): 1221-1227,1997 have described the displacement of the glycine of N-replacement to the P2 Pro residue of boric acid tripeptide thrombin inhibitors, also see US 5,585,360 described (deNanteuil et al).

It is the peptide mimics of aryl-joint-Boro (Aa) that Amparo (WO 96/20698 and patent families comprise US 5698538) has disclosed structure, and wherein Boro (Aa) can be aminoboronic acid residue, for example a BoroMpg with non-basic side chain.Described joint is that chemical formula is-(CH ₂) compound or its analogue of mCONR-(wherein m is 0-8, and R is H or some organic group), wherein peptide bond-CONR-by-CSNR-,-SO ₂NR-,-CO ₂-,-C (S) O-or-SO ₂The O-displacement.Aryl is phenyl, naphthyl or the xenyl that is selected from the group replacement of a special groups by 1,2 or 3.The most typically, these compounds are that structure is aryl-(CH ₂) n-CONH-CHR ²-BY ¹Y ²Compound, R wherein ²For example be above-mentioned neutral side chain, n is 0 or 1.

Non-peptide boric acid ester has been proposed in the detergent compositions as proteinase inhibitor.WO92/19707 and WO 95/12655 have reported that aryl-boric acid ester can be used as proteinase inhibitor in detergent compositions.WO 92/19707 has disclosed by hydrogen bonded group especially kharophen (NHCOCH ₃), sulfonamido (sufonamido) (NHSO ₂CH ₃) and the compound of alkylamino substituted boracic acid foundation group.The compound that WO 95/12655 instruction normotopia replaces is excellent.

The boric acid ester enzyme inhibitors has widespread use, forms inhibitor from stain remover to microbial spores again to pharmaceuticals.In the pharmacopedics field, have and describe for example patent documentation of the boric acid ester inhibitor of zymoplasm, factor Xa, kallikrein, elastoser, plasmin and other serine protease such as prolyl endopeptidase and Ig AI proteolytic enzyme of serine protease.Zymoplasm is a proteolytic enzyme last in the coagulation pathway, and effect is four kinds of little peptides of hydrolysis from fibrinogenic each molecule, therefore makes its polymerization site go protection.The linear fibre protein polymer is in case formation just can be by crosslinked by the factor XIIIa of thrombin activation.In addition, zymoplasm is hematoblastic powerful activator, the special receptor of thrombin action on thrombocyte.Zymoplasm is also strengthened himself by incitant V and VIII and is produced.

Other aminoboronic acid ester of serine protease or boric acid inhibitor peptides or substrate are as described in the following document:

·US 4935493

·EP 341661

·WO 94/25049

·WO 95/09859

·WO 96/12499

·WO 96/20689

·Lee S-L et al，Biochemistry 36：13180-13186，1997

·Dominguez C et al，Bioorg.Med.Chem.Lett.7：79-84，1997

·EP 471651

·WO 94/20526

·WO 95/20603

·WO97/05161

·US 4450105

·US 5106948

·US 5169841.

The peptide boric acid inhibitor of hcv protease is described in WO 01/02424.

Matteson D S Chem.Rev.89:1535-1551,1989 have looked back α-halogen boric acid ester (halo boronic esters) application in synthesizing amino boric acid and derivative thereof as intermediate and other material.Matteson has described the application of tetramethyl ethylene ketone boric acid ester in achirality is synthetic, and the application in chirality control of pinane glycol (pinanediol) boric acid ester, is included in the synthesizing of amino and amido boric acid ester.

Contreras et al J.Organomet.Chem.246:213-217,1983 have described by the ring-type boric acid ester has been carried out spectroscopy feasibility study intramolecularly N → B coordination, and described ring-type boric acid ester is to pass through Me ₂CHCMe ₂-BH ₂Prepare with the reaction of diethanolamine.

Shown that boric acid and ester cpds can be used as the inhibitor of proteasome, proteasome is a multiple catalytic proteolytic enzyme of being responsible for most intracellular protein turnovers.Ciechanover, Cell, 79:13-21,1994 instruction proteasomes are proteolysis compositions of ubiquitin-proteasome pathway, wherein protein directed degraded by puting together in a plurality of molecules of ubiquitin.Ciechanover also instructs ubiquitin-proteasome pathway to play an important role in multiple important physical process.

Adams etc., US Patent No 5780454 (1998), US Patent No 6066730 (2000), US Patent No 6083903 (2000) and the WO 96/13266 that is equal to, and US Patent No 6297217 (2001) has been described peptide boric acid ester and acid compound as proteasome inhibitor.These documents have also been described boric acid ester and acid compound and have been reduced the myoprotein degradation speed, reduce NF-kB activity in the cell, reduce the proteic degradation speed of p53 in the cell, suppress cyclin degraded in the cell, the anticancer growth, suppressing antigen presentation in the cell, suppress NF-κ B dependent cell and adhere to and suppress the application of HIV in duplicating.The WO 98/35691 instruction proteasome inhibitor of Brand etc. comprises the infraction that boronic acid compounds is used for the treatment of infraction as takes place during apoplexy or myocardial infarction.The WO 99/15183 instruction proteasome inhibitor of Elliott etc. is used for the treatment of inflammation and autoimmune disease.

Unfortunately, organic boronic relatively is difficult to obtain to analyze pure form.Therefore, alkylboronic acids and boroxin thereof air-sensitive normally.Korcek et al, J.Chem.Soc.PerkinTrans.2:242,1972, the instruction butyl boron dihydroxide is easy to oxidation in air and produces 1-butanols and boric acid.

The derivative of known boric acid provides the oxidation resistance as cyclic ester.Martichonok V etal J.Am.Chem.Soc.118:950-958 for example, 1996, set forth the diethanolamine derivatize provide protection at possible boric acid oxidation is provided.US Patent No 5,681,978 (Matteson DSet al) instruction 1,2-glycol and 1, the 3-glycol, for example tetramethyl ethylene ketone forms the stable ring-type boric acid ester that is not easy to oxidation.

Wu et al, J.Pharm.Sci., 89:758-765,2000, having discussed a kind of carcinostatic agent is the stability of compound N-(2-pyrazine) carbonyl-Phe-Leu boric acid (LDP-341 is also referred to as bortezomib).Described that " during [LDP-341] of preparation parenterai administration, described compound illustrates " irregular stable behavior ".Studied degradation pathway and inferred that described degraded is an oxidisability, initial oxidation is owing to superoxide or oxygen molecule and free radical thereof.

WO 02/059131 has disclosed stable boric acid product.Especially, these products are some boric acid peptide and/or boric acid peptide mimics, and wherein boric acid base group has been used sugared derivatize.The sugar derivatives that is disclosed has the hydrophobic amino acid side chain, and its chemical formula is as follows:

Wherein:

P is hydrogen or an amino group blocking group;

The R hydrogen or alkyl;

A is 0,1 or 2;

R ¹, R ²And R ³Be independently hydrogen, alkyl, cycloalkyl, aryl or-CH ₂-R ⁵

R ⁵All are aryl in each case, aralkyl, alkaryl, cycloalkyl, heterocyclic radical, heteroaryl or-W-R ⁶One of, wherein W is a sulfur family element and R ⁶It is alkyl;

R ¹, R ², R ³Or R ⁵Described in the cyclic group of any aryl, aralkyl, alkaryl, cycloalkyl, heterocyclic radical or heteroaryl can be optionally substituted; And

Z ¹And Z ²Form a group derived from sugar together, the atom that wherein all is attached to boron in each case is a Sauerstoffatom.

The compound of some announcements is sugar derivativess (seeing above-mentioned) of LDP-341.

Many medicines are included as a kind of active group of carboxylic acid.Have many differently between carboxylic acid and boric acid, its effect to (for example) drug conveying, stability and transhipment is not also studied.Characteristics of trivalent boron compound are that the boron atom is sp ²Hydridization, it has stayed an empty 2p on the boron atom _zTrack.BX ₃Therefore the molecule of type can be used as electron pair receptor (acceptor) or Lewis acid.Can use empty 2p _zTrack obtains the electronics of a pair of nonbonding to form covalent linkage from Lewis acid.BF ₃Therefore with Louis's base such as NH ₃Reaction forms the acid-alkali mixture, and wherein all atoms all have valence electron filling shell.

Therefore, boric acid can be used as Lewis acid, accepts OH ^-:

B(OH) ₃+H ₂O→B(OH) ₄ ^-+H ⁺

In addition, RB (OH) ₂The boric acid of type is bibasic, and has two pKa.The bond distance of the on the other hand boron of difference boron compound is very short, may be because three factors:

1.p the formation of π-p π key;

2. ion covalency resonance;

3. the reduction that repels each other between the nonbonding electronics.

The predetermined balance of KOH aqueous solution mesoboric acid and carboxylic acid is as follows (gets rid of RBO ₂ ^2-Formation):

Thrombosis

Hemostasis is the normal physiologic situation of blood, and wherein there is running balance in its composition.When described balance was destroyed, for example behind blood vessel injury, some bio-chemical pathway was triggered in this example, caused the formation (solidifying) owing to blood clot to be stopped blooding.Solidifying is a kind of dynamic and complicated process, and wherein proteolytic enzyme such as zymoplasm play a crucial role.Blood coagulation can take place by any of two cascade activations of zymogen, promptly exogenous and intrinsic coagulation cascade approach.Factor VIIa in the extrinsic pathway and the factors IX a in the intrinsic pathway are the important determinatives that factor X is activated as Xa, Xa autocatalysis thrombogen activates and is zymoplasm, and while zymoplasm catalysis fibre proteinogen monomer polymerization successively is a fibrin polymer.Therefore proteolytic enzyme last in each approach be zymoplasm, and its effect is the 4 kind little peptides (two kind FpAs and two kind FpBs) of hydrolysis from each fibrinogen molecule, makes its polymerization site go protection thus.In case the linear fibre protein polymer forms, then it can be by self by the factor XIIIa of thrombin activation and crosslinked.In addition, zymoplasm is a kind of effective platelet activating agent, and it works to special receptor on this.Hematoblastic thrombin activation causes cell aggregation and extra factor secretion, further promotes the generation of hemostatic plug.Zymoplasm is also strengthened himself producing by incitant V and VIII and (is seen Hemker and Beguin:Jolles, et.al., " Biology and Pathology ofPlatelet Vessel Wall Interactions; " pp.219-26 (1986), Crawford and Scrutton:Bloom and Thomas, " Haemostasis and Thrombosis; " pp.47-77, (1987), Bevers, et.al., Eur.J.Biochem.122:429-36,1982, Mann, Trends Biochem.Sci.12:229-33,1987).

Proteolytic enzyme is the enzyme in special peptide bond crack protein matter.Cuypers et al., J.Biol.Chem.257:7086,1982 and the reference quoted, proteolytic enzyme is fallen into 5 types according to the mechanistic view of life principle: Serine, cysteinyl or mercaptan, acid or aspartyl, Threonine and metalloprotease.The member of each classification has similar avtive spot amino-acid residue all by the hydrolysis of similar machine-processed catalysis peptide bond, and to classification specific inhibitor sensitivity.For example, all serine proteases of having identified all have an avtive spot serine residue.

Solidifying egg white enzyme zymoplasm, factor Xa, factor VIIa and factors IX a are that the cracking to sequence-specific Arg-Xxx peptide bond has the specific serine protease of trypsin-like.As other serine protease, the cracking incident at first is to attack avtive spot Serine on the key of easy fracture of substrate, causes the tetrahedron intermediate to form.This tetrahedron intermediate N-terminal of collapsing and forming acyl enzyme and discharge the cracked sequence subsequently.The hydrolysis of acyl enzyme discharges C-terminal then.

As mentioned above, thrombocyte plays two kinds of vital role in normal haemostasis.At first, formed initial hemostatic plug by hematoblastic gathering, it reduces effusive blood from the destructive blood vessel immediately.Secondly, platelet surface can become active and strengthen blood coagulation, and this is a kind of character that is called the thrombocyte procoagulant activity.This can be used as and has factor Va and Ca ²⁺Situation under, the speed that factor Xa activates thrombogen improves and observes, and is called the thrombogen enzyme reaction.Normally, almost there is not (also is seldom even if having) coagulation factors on the platelet surface of Ci Jiing, but when thrombocyte is activated, usually become availablely at the phosphatide (phosphatidylserine and phosphatidylinositols) of the negative charge of the kytoplasm side of film, and provide the surface that two steps solidified takes place thereon.Phosphatide on the activated platelet surface extremely promotes to cause the reaction of zymoplasm formation, so that zymoplasm can be compared quicker generation with its neutralization by Antithrombin III.Having or do not having in the heparin situation, the reaction that takes place on platelet surface is not easy to be suppressed (See Kelton and Hirsch:Bloom andThomas by anticoagulative substance natural in the blood such as Antithrombin III, " Haemostasis and Thrombosis, " pp.737-760, (1981); Mustard etal:Bloom and Thomas, " Haemostasis and Thrombosis, " pp.503526, (1981); Goodwin et al; Biochem.J.308:15-21,1995).

Can think that thrombus is a kind of unusual product of normal mechanism, and can be interpreted as a kind of block or the settling that for example from blood constitutent, form on the surface of the heart or blood vessel at cardiovascular systems.Thrombosis is considered to pathological condition, and wherein incorrect hemostatic mechanism activity causes thrombosis in the blood vessel.Discerned the thrombus of three kinds of base types now:

White thrombus, it sees artery usually, mainly is made up of thrombocyte;

Red thrombus, it sees vein, mainly is made up of scleroproein and red corpuscle;

Mixed thrombus, it is become to be grouped into by white thrombus and red thrombus.

The composition of thrombus is subjected to form at it influence of the blood flow rate at position.The thrombus of common white thrombocyte enrichment forms in the fast system of blood flow rate, and the red thrombus that solidifies forms in the zone of being retarded by silt.The high shear rate of artery prevents that the intermediate of blood coagulation from accumulating on the round-robin artery sidewall: have only thrombocyte to have by the ability of Willebrand factor formation with damage location bonded thrombus.Only this thrombus of being made up of thrombocyte is instability and dispersive.If stimulate by force then thrombus will form once more, continue then to disperse to reduce until stimulating.For making thrombus stable, must form scleroproein.Aspect this, a spot of zymoplasm can accumulate in the platelet thrombus and factor Va is activated and the stimulating platelet procoagulant activity.These two incidents cause factor Xa to improve 300,000 times to the rate of activation of thrombogen is whole.The fibrin deposition thing makes platelet thrombus stable.Indirect thrombin inhibitors such as heparin are suppressing not have clinical efficacy aspect the stimulating platelet procoagulant activity.Therefore, the therapeutical agent of inhibition thrombocyte procoagulant activity will be useful on treatment or prevention of arterial pathological change of thrombus.

In the round-robin venous side, thrombus comprises scleroproein: because venous side slow blood flow so zymoplasm can be assembled, thrombocyte only plays a secondary role.

Therefore do not think that thrombosis is a single indication, but comprise a class indication of the different subclass that are fit to different therapeutical agents and/or scheme.Therefore, for example dvt formation of disease, cerebrovascular artery thrombosis and the pulmonary infarction index as the approval medicine will be treated by administration.Two kinds of main thrombosis subclass are artery thrombosis and venous thrombosis.Artery thrombosis comprises special pathology such as acute coronary syndrome ((for example Acute Myocardial Infarction (heart attack that is caused by Coronary thrombosis)), cerebrovascular artery thrombosis (apoplexy that is caused by thrombosis in the cerebrovascular Arterial system) and peripheral arterial thrombosis.The pathology that is caused by venous thrombosis for example is that dvt forms and pulmonary infarction.

Thrombotic processing generally comprises uses antiplatelet drug (inhibitor of platelet aggregation) to control further thrombosis, reach and use the new clot that forms of thrombolytic agent dissolving, one or both of these two kinds of preparations can be united or applied in any combination with anti-coagulant.Also can preventative use anti-coagulant in the thrombotic patient of easy trouble is thought in treatment.

At present, clinical the most effective two class medicines as anti-coagulant are heparin and vitamin K antagonist.Heparin is the not clear mixture that combines and therefore strengthen the sulfated polysaccharide of its effect with Antithrombin III.Antithrombin III is the inhibitor (seeing Jaques, Pharmacol.Rev.31:99-166,1980) of a kind of natural generation of activated factor IXa, Xa, XIa, zymoplasm and possible XIIa.The vitamin K antagonist, the warfarin of for example knowing is by carboxylation behind the rrna that suppresses vitamin K-dependent clotting factor II, VII, IX and X work indirectly (seeing Hirsch, Semin.Thromb.Hemostasis 12:1-11,1986).Although heparin and vitamin K antagonist are the thrombotic effective ways of treatment, but it has the disadvantageous side effects such as (in the situations of heparin) of hemorrhage, heparin-induced thrombopenia, and mutability between tangible patient, it is little and unpredictable to cause treating security.

Use the zymoplasm of the blood coagulation system that directly works and the inhibitor expectation of other serine protease and can alleviate these problems.For this reason, after tested a large amount of serpins, comprise the boric acid peptide, promptly contain the peptide of the boric acid analogue of a-amino acid.Discussed the boric acid thrombin inhibitors that directly works at the preamble of this specification sheets, chapters and sections further describe once.

Neutral P1 residue boric acid peptide thrombin inhibitors

(WO 92/07869 and patent families comprise US 5648338) such as Claeson etc. (US 5574014 etc.) and Kakkar disclosed and had for example lipotropy thrombin inhibitors of an alkoxyalkyl side chain of a neutrality (uncharged) C-terminal (P1) side chain.

Patents of the same clan such as Claeson etc. and Kakkar have disclosed and have contained aminoacid sequence D-Phe-Pro-BoroMpg[(R)-Phe-Pro-BoroMpg] boric acid ester, it is the high degree of specificity inhibitor of zymoplasm.Particularly Cbz-(the R)-Phe-Pro-BoroMpg-O tetramethyl ethylene ketone (being also referred to as TRI 50b) that can mention in these compounds.Corresponding free boric acid is called TRI 50c.About the further information of TRI 50b and related compound, the reader can consult following document:

·Elgendy S et al.，in The Design of Synthetic inhibitors of Thrombin，Claeson G et al Eds，Advances in Experimental Medicine，340：173-178，1993.

·Claeson G et al，Biochem J.290：309-312，1993

·Tapparelli C et al，J Biol Chem，268：4734-4741，1993

·Claeson G，in The Design of Synthetic inhibitors of Thrombin，Claeson G et al Eds，Advances in Experimental Medicine，340：83-91，1993

·Phillip et al，in The Design of Synthetic inhibitors of Thrombin，Claeson G et al Eds，Advances in Experimental Medicine，340：67-77，1993

·Tapparelli C et al，Trends Pharmacol.Sci.14：366-376，1993

·Claeson G，Blood Coagulation and Fibrinolysis 5：411-436，1994

·Elgendy et al，Tetrahedron 50：3803-3812，1994

·Deadman J et al，J.Enzyme Inhibition 9：29-41，1995

·Deadman J et al，J.Medicinal Chemistry 38：1511-1522，1995.

The tripeptide sequence of TRI 50b has 3 chiral centres.Think that at least the Phe residue is that (R)-configuration and Pro residue are natural (S)-configurations in the compound with commercial useful inhibitor activity; In isomer, be sure of that the Mpg residue is (R)-configuration with the useful inhibitor activity of commerce.Therefore, think that the TRI 50b steric isomer of activity or maximum activity is R, S, the R configuration, can following chemical formula shown in:

Though having been found that the thrombin inhibitors that works indirectly is used for the treatment of easy trouble or suffers from the patient of venous thrombosis, but arterial thrombus is not such, increases manyfold with treatment (prevention) arterial thrombus because need to be used for the treatment of venothrombotic dosage.The dosage of this increase typically causes bleeding, and this makes the thrombin inhibitors that works indirectly be unsuitable for or not be preferred for treating arterial thrombus.Heparin and low-molecular-weight derivative thereof are indirect thrombin inhibitorss, therefore are unsuitable for treating arterial thrombus.Oral direct thrombin inhibitors is a progress of treatment arterial thrombus, but therapeutic index is lower than desirable therapeutic index (therapeutic indices), promptly has the hemorrhage situation that is higher than level of hope under therapeutic dose.

Many organic boronic compounds can be divided into lipophilic class or hydrophobic class.Typically, this compound comprises following compound:

Whole or Most amino-acids is hydrophobic boric acid peptide,

At least half amino acid is hydrophobic and has the boric acid peptide of a hydrophobicity N-terminal substituting group (amino protecting group),

Non-peptide based on hydrophobic group.

Oral absorption

Can in gi tract, absorb by active or passive approach.That active absorption by transporting mechanism is tending towards between individuality is variable and relevant with intestinal contents (Gustafsson et al, Thrombosis Research, 2001,101,171-181).The enteron aisle of having determined top is the main position that oral pharmaceutical absorb.Especially, duodenum absorbs the position, convention target bonding part of oral pharmaceutical because its surface-area is big.Intestinal mucosa is controlled transcellular passive absorption as a barrier: ionic absorb be blocked and help the lipophilic quasi-molecule stride cell absorb (Palm K et al., J.Pharmacol and Exp.Therapeutics, 1999,291,435-443).

Oral pharmaceutical need continue and fully absorb.Disagreeable is between the individuality and the difference between the different opportunitys of same individuality and absorb difference.Similarly, the medicine that general unacceptable bioavailability level is low (only the promoting agent that gives of small part is absorbed).

Passively be absorbed with the absorption that is beneficial to non-ionic compound, this is a kind of approach relevant with unchangeability, therefore is preferably to continue absorption.Passive mechanism of absorption is particularly conducive to lipophilic class material, therefore shows to be beneficial to continuing and the height oral absorption of non-ionic lipophilic class medicine most.

The needed typical functionality of the interaction of medicine and its physiology target position is functional type group such as carboxylic acid and sulfonic acid.These groups (at pH2-3) under one's belt exist with protonated form, but in the intestinal juice of higher pH ionization are to a certain degree arranged.Being used to avoid a strategy of carboxylate salt or sulfonate ionization is that it is existed with ester-formin, in case be absorbed in the lumen of vessels then cleaved.

For example, do not reach the direct acting thrombin inhibitors melagatran of best gastrointestinal absorption, have terminal carboxyl(group) and amidino groups group, and when described carboxylic acid and amidino groups group be pure zwitter-ion when all being charged at pH 8-10.Therefore developed a kind of prodrug H376/95, it has the protecting group of carboxylic acid and amidine, and is than the more lipophilic molecule of melagatran.Described prodrug is higher than 80 times of melagatran through the epithelial permeability coefficient of cultivating of Caco-2, oral administration biaavailability is higher than 2.7-5.5 times of melagatran, and less (the Gustafsson et al of mutability in the scope under drug plasma concentration vs time curve, Thrombosis Research, 2001,101,171-181).

The oral absorption of boric acid peptide, boric acid peptide mimics and other organic boric acid ester

The boric acid ester group of TRI 50b is cracking rapidly under the condition of blood plasma, forms corresponding boric acid base group, and this group is considered to the active group of the catalytic site of Trombin inhibiting.

Boric acid is the functional group of divalence, has the boron-oxygen key length (1.6 of more typical singly-bound characteristics

), different with the key of C-O in carboxylic acid and the sulfonic acid and S-O.Therefore, boric acid base group has two kinds of ionization potentiality.Boric acid base group is in the pH lower section ionization of duodenal juice, and is unbecoming with the passive duodenum absorption of hope.Therefore, a kind of charged boric acid ester inhibitor H-D-PheProBoroArg be by main active transport mechanism absorbed (Saitoh, H.andAungst, B.J., Pharm.Res., 1999,16,1786-1789).

The peptide boric acid (this acid is called as TRI 50c) that this cracking by TRI 50b forms can not dissolve in water relatively, particularly under acidity or neutral pH, and is tending towards being difficult in stomach and duodenum absorbing.This sour structure is Cbz-Phe-Pro-BoroMpg-OH.

And peptide boric acid Cbz-Phe-Pro-BoroMpg-OH reaches such degree and is unfavorable for passive transport in the lower section ionization of duodenum condition, and this sour ester is designed to high speed passive transport (therefore consistent).Tripeptide sequence Phe-Pro-Mpg has a non-alkaline P1 side chain (particularly methoxy-propyl), and described thus tripeptides is made up of three nonpolar amino acids.The ester of described peptide boric acid is non-ionizable, and ester forms kind and further authorize lipotropy character, promotes the high speed passive transport thus.

But other diol ester of the verified TRI of supposition 50b of computing technique and Cbz-Phe-Pro-BoroMpg-OH has good biology availability.Therefore, polar surfaces zone (PSAd) is a parameter inferring bioavailability, is higher than 60

The PSAd value and the bioavailability of passive transcellular transport and known drug closely related (Kelder, J.Pharm.Res., 1999,16,1514-1519).The diol ester of above-mentioned peptide boric acid is comprised the mensuration of pinacol ester TRI 50b, this diol ester is shown has and be higher than 60 The PSAd value, infer passive transport and good bioavailability, as shown in table 1:

Table 1: the PSAd value of the Cbz-Phe-Pro-BoroMpg-OH diol ester of selection

Glycol	The PSAd value
Glycol	The PSAd value	Tetramethyl ethylene ketone	98.74
The pinane glycol	90.64	Tetramethyl ethylene ketone	98.74

It is too unstable that corresponding single hydroxyl alcohol (for example alkanol) ester is considered to, and discharges acid in external natural cracking.The ester of glycol such as pinane two pure and mild tetramethyl ethylene ketones has with the ester of single hydroxyl alcohol compares the enhanced kinetic stability, and after partial hydrolysis was monoester derivates, they were tending towards reconfiguring by intramolecular reaction easily.

Summary of the invention

Be these features that highly need of balance TRI 50b, disclosed TRI 50b and be tending towards hydrolysis.Therefore under the acidic conditions that HPLC analyzes, TRI 50b is changed into short sour form of transformation period, this means at duodenum and in gi tract, be hydrolyzed to ionic species in other position potential duodenum, ionic species will be resisted passive transport, if and words are arranged and so on, then absorb, show indefinite bioavailability at most by active transport.

TRI 50b is to preparation and the preparation thereof at compound of the unstable of hydrolysis, and contains in its storage of pharmaceutical preparation and also have the potential disadvantage.

The another kind of challenging difficulty that is caused by TRI 50b is that data illustrate the noticeable change in the bioavailability between the object.This mutability can produce a kind of unacceptable drug candidates, therefore needs to reduce this mutability that observes.

Solving the instable a kind of Perfected process of TRI 50b is to develop the more stable diol ester of hydrolysis, because this diol ester is similar to TRI 50b, can predict that comparing it with TRI 50c has the oxidation resistance.In this regard, the big I of known ring influences the stability of boric acid ester, and glycolato boron has been shown compares with tetramethyl ethylene ketone and have enhanced water-based stability (D.S.Matteson, Stereodirected Synthesis with Organoboranes, Springer-Verlag, 1995, ch.1).Similarly, the pinane diol ester is compared more stable with tetramethyl ethylene ketone; Be sure of that this is because pinane glycol group is the height sterically hindered and is unfavorable for nucleophillic attack to boron.In fact, reported transesterify (Brosz, CS, Tet.Assym, 8:1435-1440,1997), and reverse procedure is not smooth from tetramethyl ethylene ketone to the pinane glycol.To such an extent as to can not cracking yet think that the pinane diol ester is too inactive in blood plasma, therefore a kind of diol ester of improvement need be provided.

The instable another kind of method that solves TRI 50b is to give TRI 50c in its original position.Yet mutability is too high in the bioavailability of TRI 50c Notes of Key Data TRI 50c.

TRI 50c also has unstable, because there is the trend of boric acid peptide moiety self degraded by going boronation (carbon-boron bond cracking), the oxidative pathway by previous teach literature carries out (for example Wu et al, as above-mentioned).Degradation Level can be very high.

The above-mentioned character of TRI 50b and TRI 50c is non-to be limited to this compound, but total by other boric acid peptide ester and acid, even the character of this other boric acid peptide is quantitatively different.

The present invention shows enhanced stability etc. with some organic boronic product to be found to be the basis.

Benefit of the present invention comprises the especially instable problem of TRI 50b of glycol borate ester that solved, that is to say that the product that the present invention discloses especially provides pharmaceutical active compounds, its with TRI 50b and other can correlated ester aspect hydrolysis stable, compare more stable.The present invention has further comprised the instable problem of solution organic boronic, that is to say that the product that the present invention discloses especially provides pharmaceutical active compounds, and it is more stable that itself and TRI 50c compare boronation.The stability that provides in framework of the present invention is not absolute, improves but compare with correlated compound.The benefit that provides of the present invention further comprises the product that is provided at the unanticipated that has effectiveness in the oral preparations.

The present invention has disclosed a kind of aminoboronic acid derivative, and it has avoided the shortcoming of pinacol ester.The present invention further comprises a kind of peptide boric acid derivative, and it shows enhanced stability.Especially, the present invention includes a kind of pharmaceutical preparation, it comprises a kind of aminoboronic acid derivative and can provide a kind of ionic boric acid peptide material to avoid the shortcoming of pinacol ester simultaneously in duodenum, and bioavailability can be provided.The present invention further comprises boric acid derivatives, and it is to hydrolysis and go boronation relatively stable and useful in the oral preparations of Trombin inhibiting.

The present invention relates to the base addition salt of acceptable some the organic boronic medicine of a kind of medicine, particularly hydrophobicity boric acid peptide (for example dipeptides or tripeptides) reaches the thrombin inhibitors that more especially has non-alkaline P1 group.As a kind, this salt is not only opposite with the guidance in prior art field, and additionally has the improved stability level, and this can not explain or predict based on the known chemical principle.

On the one hand, the present invention relates to the base addition salt of the acceptable boric acid of medicine, the hydrophobic group that described boric acid has and has a neutral aminoboronic acid residue and is connected with this neutrality aminoboronic acid residue through a peptide bond, described neutral aminoboronic acid residue energy bind thrombin S1 sublocus, described hydrophobic group can bind thrombin S2 and S3 sublocus.In first embodiment, disclosed the base addition salt of boric acid shown in the acceptable for example formula of a kind of medicine (I):

Wherein

Y comprises a hydrophobic group, itself and aminoboronic acid residue-NHCH (R ⁹)-B (OH) ₂Substrate binding site to zymoplasm has affinity together; And

R ⁹Be a straight chained alkyl group that is interrupted by one or more ehter bond (for example 1 or 2), wherein the sum of oxygen and carbon atom is 3,4,5 or 6 (for example 5), perhaps R ⁹Be-(CH ₂) _m-W, wherein m is 2,3,4 or 5 (for example 4), W is-OH or halogen (F, Cl, Br or I).R ⁹In a hypotype of compound is alkoxy-alkyl group, for example contains the alkoxyalkyl of 4 carbon atoms.

The present invention comprises the base addition salt of the hydrophobicity boric acid inhibitor of the acceptable zymoplasm of medicine, and therefore comprises this salt of peptide boric acid, and it represents to be higher than 1.0 in physiological pH and at the partition ratio between the hot alcohol and water of 1-n-under 25 ℃ of conditions with log P.The partition ratio of some peptide boric acids among the present invention is at least 1.5.The partition ratio of a class hydrophobic peptide boric acid is for being no more than 5 among the present invention.

As some example announcement is the base addition salt of the hydrophobicity boric acid inhibitor of the acceptable zymoplasm of medicine.This inhibitor can contain hydrophobic amino acid, and this amino acid comprises that side chain is alkyl, contains oxygen in the chain and/or the alkyl that is connected with the rest part of molecule by oxygen in the chain or heteroaryl or by the amino acid of any aforementioned group of hydroxyl, halogen or trifluoromethyl replacement.Aforementioned group, aryl, heteroaryl that representative hydrophobic side chain comprises alkyl, alkoxyalkyl, replaced by at least a aryl or heteroaryl, aryl that replaces by at least a alkyl and the heteroaryl that replaces by at least a alkyl.Proline(Pro) and ring do not have substituted or carry out cyclosubstituted imino-acid by the listed a kind of group of last sentence also is hydrophobic.

Some hydrophobic side chains contain 1-20 carbon atom, for example have the non-annularity group of 1,2,3 or 4 carbon atom.The side chain that comprises a cyclic group is phenyl or the alkyl that replaced by one or two phenyl in many cases typically but the nonessential 5-13 of a containing ring members.

The non-peptide of hydrophobicity is typically based on the above-mentioned group that can form the hydrophobic amino acid side chain.

Hydrophobic compound for example can contain an amino group and/or acid groups (for example-COOH ,-B (OH) ₂).Normally, they do not contain the multiple polar group of arbitrary type.

One class hydrophobicity organic boronic is in physiological pH and under 25 ℃ of conditions, and the partition ratio between the hot alcohol and water of 1-n-represents to be higher than 1 with log P.For example, the partition ratio of TRI 50c is about 2.

Some subclass of hydrophobicity organic boronic be hereinafter title in " some embodiment describe in detail " by formula (I) with those compounds (III).

As another embodiment announcement is the base addition salt of peptide boric acid shown in the acceptable formula of medicine (II):

Wherein:

X is and N-terminal amino group bonded group, can be that H is to form NH ₂The character of X is not crucial, can be above-mentioned one special X group.In one embodiment, be the benzyloxycarbonyl of mentioning.

Aa ¹Be the amino acid with an alkyl side chain, it contains and is no more than 20 carbon atoms (for example maximum 15 and optional maximum 13 carbon atoms) and comprises at least one cyclic group with maximum 13 carbon atoms.In certain embodiments, aa ¹Cyclic group 5 or 6 ring memberses are arranged.For example, aa ¹Cyclic group can be aromatic yl group, particularly phenyl.Typically, at aa ¹One or two cyclic group is arranged in the side chain.Some side chain comprises or by being formed by one or two 5 or 6 member's cyclosubstituted methyl.

More particularly, aa ¹It is Phe, Dpa or its hydrogenant analogue wholly or in part.Therefore described complete hydrogenant analogue is Cha, the present invention includes the salt medicine of metal-salt for example that comprises organic boronic, and described organic boronic is a thrombin inhibitors, particularly has the selective thrombin inhibitors of a neutral P1 (S1 in conjunction with) group.About the S3 of bind thrombin, the information of the group in S2 and S1 site is seen for example Tapparelli C et al, Trends Pharmacol.Sci.14:366-376,1993; Sanderson P et al, Current Medicinal Chemistry, 5:289-304,1998; RewinkelJ et al, Current Pharmaceutical Design, 5:1043-1075,1999; And Coburn C Exp.Opin.Ther.Patents 11 (5): 721-738,2001.Zymoplasm inhibition salt of the present invention is non-be limited in the publication that last sentence is listed, describe have S3, those of S2 and S1 affinity groups.

Aa ²It is the imino-acid that 4-6 ring members arranged.Perhaps, aa ²By C ₃-C ₁₃Hydrocarbyl group for example comprises C ₃-C ₆The C of alkyl ring ₃-C ₈The Gly that hydrocarbyl group replaces at N-terminal; Described hydrocarbyl group can be saturated, and for example the N-terminal substituting group is cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.The hydrocarbyl group that contains one or more unsaturated link(age) can be described phenyl and the methyl or the ethyl that are replaced by phenyl, for example 2-styroyl, and β, β-dialkyl benzene ethyl.

Have in the document arguement part is whether aqueous solution mesoboric acid root forms " trilateral " B (OH) ₂Perhaps " tetrahedroid " B (OH) ₃-boron class, but as if the NMR sign show first pKa part that is lower than boric acid at pH, and main boron class is neutral B (OH) ₂In duodenum, 6 and 7 between, therefore there probably preponderate probably by leg-of-mutton kind for pH.In any situation, symbol-B (OH) ₂Comprise tetrahedron and trilateral boron class, and represent that in this manual the symbol of trilateral boron class also comprises the classification of tetrahedroid.This symbol further can comprise the boric acid base group (boronic group) of acid anhydride form.

Described salt can be solvate form thereof, particularly hydrate forms.

Described salt can comprise or be made up of acid salt substantially, and its mesoboric acid is independent deprotonation.Therefore the present invention includes and have the consistent stoichiometric product of metal/borate of product mesoboric acid foundation group that carries single negative charge with main (more than the 50mol%).

The present invention also provides the oral preparations of described salt.Especially, the invention provides the oral preparations that comprises the described salt of solid phase, for example the salt with particulate is mixed with the tablet of compression or is mixed with capsule.

According to a further aspect in the invention, the method that provides a kind of treatment wherein to need the illness of anti-thrombosis activity, described method comprise the salt of suffering from or being in boric acid shown in the formula (I) of the patient's oral administration significant quantity among this illness danger.

As hereinafter further describing, the present invention also provides the haemodialysis solutions that comprises salt of the present invention.

Salt of the present invention comprises by boric acid and highly basic and reacts obtainable product (characteristic with the product that obtains by this reaction), understands term of the present invention " salt " thus.Therefore contain discrete positively charged ion and negatively charged ion at the nonessential product that means of the term " salt " of the product that is disclosed, and be interpreted as comprising the obtainable product of reaction that uses boric acid and alkali.The present invention comprises the product of coordination compound form on greater or lesser degree.Therefore the present invention also provides by boric acid (I) and highly basic and has reacted obtainable product (having the characteristic by the obtainable product of this reaction), and the treatment of this product, comprises prophylactic applications.

The present invention is not subjected to the preparation method's of salt restriction, as long as they contain borate class and a counterion derived from boric acid (I).This borate class can be the borate anion of its any equilibrium form.Term " equilibrium form " is meant the multi-form of same compound, its can in the balance equation, represent (for example with the boron trioxide balance and with the balance of different borate ions in boric acid).The borate of solid phase can form acid anhydride, and works as when the borate that is disclosed is solid phase and can comprise boron trioxide, as boric acid balance kind.Nonessential alkali and the described salt of boric acid (I) prepared in reaction by containing counterion.In addition, the present invention includes such salt product, it is considered to prepare indirectly by this acid/alkali reaction, and by the obtainable salt of this indirect preparation (characteristic with the product that obtains by this reaction).Possible indirect preparation method for example can be such method, wherein after the recovery of initial salt, is purified and/or handles to modify its physicochemical property, for example modifies its solid form or hydrate forms or the two.

In some embodiments, the positively charged ion of described salt is a univalent.

In some embodiments, described salt comprises the acid anhydride class, and in other embodiments, they are no acid anhydride class substantially.

Described salt can be isolating form.Determine that as method described salt can have for example about at least 90% purity, for example is greater than or equal to about 95% by embodiment 28.In the situation of pharmaceutical preparation, the form of this salt can with medicine acceptable diluent, vehicle or carrier combinations.

The present invention includes a kind of from the method for corresponding boric acid as intermediate preparation salt, and intermediate boric acid shown in the formula (I) and its method of preparation.

Others of the present invention and embodiment are set forth in the following description and claims.

In the description and claim of this specification sheets, term " comprises " and " containing " and variation term thereof, for example " comprises ", is meant " comprising but non-being limited to ", and does not get rid of other group, additive, composition, integer or step.

The data that the present patent application contains show organic boronic stability (going the boronation resistance) can by with its with salt for example the form of metal-salt provide and increase.In the discrete experiment, the ammonium salt of TRI50c shows as based on drying and decomposes generation ammonia, and choline salt shows as and is decomposed into the boronation mishmash rapidly simultaneously.Although experimentized reappearing these unrepeatable observed results, but a subclass be provided, wherein got rid of ammonium salt and choline salt.Described salt can be acid salt.In any situation, this stabilization technology has formed a part of the present invention, and the organic boronic of for example describing down applicable to " background " title reaches the organic boronic of describing in the publication of mentioning under this title.

The accompanying drawing summary

Fig. 1 is the synoptic diagram of embodiment 32, is illustrated in mouth phase clearance rate and kinetics after p.o. gives TRI 50b or TRI 50c.

Fig. 2 is another synoptic diagram of embodiment 32, is illustrated in the mouth phase clearance rate and the kinetics that give in duodenum behind TRI 50b or the TRI 50c.

The detailed description of some embodiment

Nomenclature

Use following term and abbreviation in this manual:

Be meant its leg-of-mutton acid groups-B (OH) at the used term of the salt of boric acid " acid salt " ₂One-OH group by a kind of salt of deprotonation.Therefore wherein the borate group carries a negative charge and can be expressed as-B (OH) (O ^-) or [B (OH) ₃] ^-Salt be acid salt.The cationic salts with valency n contained in this term, and its mesoboric acid and cationic molar ratio are about n: 1.In practice, the stoichiometry of observation can not really be n: 1, but with the n of theory: 1 stoichiometry is consistent.For example, the positively charged ion quality of observation can be at n: change being no more than approximately 10% on the quality that 1 stoichiometry is calculated, for example be no more than about 7.5%; In some cases, the variation between the quality of the positively charged ion quality of observation and calculating is no more than about 1%.The quality of calculating is based on the borate of triangular form.(at atomic level, the salt consistent with the stoichiometry of acid salt can contain the borate mixture of protonation state, and its mean value is close with single deprotonation, and this " mixing " salt is included in the term " acid salt ").Acid salt for example is single sodium salt and half zinc salt.

Alpha-amino boronic acid or Boro (aa) are meant wherein CO ₂Group is by BO ₂Metathetical amino acid.

Term " amino group blocking group " is meant and is used to derive especially any group of peptide or amino acid whose N-terminal amino group of an amino group.This group comprises but non-alkyl, acyl group, alkoxy carbonyl, aminocarboxyl and the alkylsulfonyl group of being limited to.Yet, non-those the special protecting groups in organic synthesis, used always that are limited to of term " amino group blocking group ", also non-being limited to, be easy to those groups of cracked.

The used phrase of the present invention " medicine is acceptable " is meant those compounds, material, composition and/or formulation, they are in rational medical judgment scope, be suitable for contacting with the tissue of human body or animal and not having too much toxicity, pungency, transformation reactions or other problem or complication, corresponding with rational benefit/risk.

Term " thrombin inhibitors " is meant a kind of product, it is in reliable pharmacology determination range, potential or really as the inhibitor of zymoplasm aspect pharmacology, comprise the material that comprises the pharmacologic activity thing and be described to recommend or authorization as thrombin inhibitors.This thrombin inhibitors can be optionally, and promptly it compares with other proteolytic enzyme in reliable pharmacology determination range that to be considered to zymoplasm be optionally; Term " selective thrombin inhibitors " comprises the material that comprises the pharmacologic activity thing and is described to be recommended or authorizes as selective thrombin inhibitors.

Term " heteroaryl " is meant a loop systems, and it has heteroatoms at least 1 (for example 1,2 or 3) ring, and has a cyclic olefinic bond system of puting together.Term " heteroatoms " comprises oxygen, sulphur and nitrogen, wherein not too preferred sometimes sulphur.

" natural amino acid " is meant amino acid whose a kind of L-amino acid (or its residue) of the positively charged and negative charge that is selected from following neutrality (hydrophobicity or polarity):

Hydrophobic amino acid

The A=Ala=L-Ala

The V=Val=Xie Ansuan

The I=Ile=Isoleucine

The L=Leu=leucine

The M=Met=methionine(Met)

The F=Phe=phenylalanine

The P=Pro=proline(Pro)

The W=Trp=tryptophane

Polarity (neutral or uncharged) amino acid

The N=Asn=l-asparagine

The C=Cys=halfcystine

The Q=Gln=glutamine

The G=Gly=glycine

The S=Ser=Serine

The T=Thr=Threonine

Y=Tyr=tyrosine

Positively charged (alkalescence) aminase

The R=Arg=arginine

The H=His=Histidine

K=Lys=Methionin

Electronegative amino acid

The D=Asp=aspartic acid

E=Glu=L-glutamic acid

The ACN=acetonitrile

Amino acid=a-amino acid

Base addition salt=in free acid (being boric acid in this situation) adds a kind of mineral alkali or organic bases and the salt for preparing

The Cbz=benzyloxycarbonyl

Cha=Cyclohexylalanine (a kind of hydrophobicity alpha-non-natural amino acid)

Charged (as be used for the fragment of medicine or drug molecule, for example amino-acid residue)=carry electric charge in physiological pH is as in the situation of amino, amidino groups or carboxylic group

Dcha=dicyclohexyl L-Ala (a kind of hydrophobicity alpha-non-natural amino acid)

Dpa=two phenylalanines (a kind of hydrophobicity alpha-non-natural amino acid)

No matter medicine=a kind of pharmacology benefit materials is activated in vivo or a kind of prodrug

The i.v.=intravenously

Mpg=3-methoxy-propyl glycine (a kind of hydrophobicity alpha-non-natural amino acid)

The valency of multivalence=at least two valencys, for example two or trivalent

Neutral (as be used for the fragment of medicine or drug molecule, for example amino-acid residue)=uncharged=at the physiological pH neutral

Pinac=tetramethyl ethylene ketone=2,3-dimethyl-2,3-butyleneglycol

Pinane glycol=2,3-pinane glycol=2,6,6-trimethylammonium two ring [3.1.1] heptane-2,3-glycol

The Pip=2-nipecotic acid

P.o.=per os=gives (so oral preparations gives by p.o.) by the oral cavity

S.c.=is subcutaneous

The sufficiently high pKb of highly basic=have with the alkali of acid reaction.The pKb of suitable this alkali is 7 or higher, for example 7.5 or higher, for example about 8 or higher.

The THF=tetrahydrofuran (THF)

The Thr=zymoplasm

Compound

Product of the present invention comprises the salt of boric acid, its have one can bind thrombin neutral aminoboronic acid residue and the energy bind thrombin S2 that is connected with this neutrality aminoboronic acid residue through a peptide bond and the hydrophobic group of S3 sublocus of S1 sublocus.The present invention comprises the salt of acid shown in the formula (I):

Wherein

R ⁹Be the straight chained alkyl group that is interrupted by one or more ehter bond, wherein the sum of oxygen and carbon atom is 3,4,5 or 6 (for example 5), perhaps R ⁹Be-(CH ₂) _mW, wherein m is 2,3,4 or 5 (for example 4), W is-OH or halogen (F, Cl, Br or I).(O-) straight chained alkyl of Zhong Duaning for example can be alkoxyalkyl (place interruption) and the alkoxy alkoxy alkyl of mentioning (interruption of two places) by one or more ehter bond.R ⁹Be an alkoxy-alkyl group in a kind of compound hypotype, for example contain the alkoxyalkyl of 4 carbon atoms.

Typically, YCO-comprises the amino-acid residue of S2 sublocus bonded (no matter natural or non-natural) with zymoplasm, and amino-acid residue is connected at N-terminal with a group of the S3 sublocus of bind thrombin.

In the acid shown in the class formula (I), YCO-is the dipeptide residue of optional N-terminal protection, and it combines with the S3 and the S2 binding site of zymoplasm, and the peptide key option in the described acid is also independently by C ₁-C ₁₃Hydrocarbyl group replaces at N-terminal, optional chain interior and/or the interior nitrogen of ring, oxygen or the sulphur and optional by the substituting group replacement that is selected from halogen, hydroxyl and trifluoromethyl of containing of described hydrocarbyl group.When having the N-terminal protecting group, it can be above-mentioned radicals X (except a hydrogen).Common described acid does not contain the peptide bond that N-terminal replaces; In the peptide bond part that exists N to replace, substituting group is the alkyl of 1C-6C normally, for example saturated alkyl; The N in some embodiments substituting group comprises a ring, cycloalkyl for example, and can be cyclopentyl for example.One class acid has a N-terminal protecting group (for example X group) and unsubstituted peptide bond.

At YCO-is a dipeptide residue part (no matter whether N-terminal is protected), and S3 can be that R configuration and/or S2 can be the S configurations in conjunction with residue in conjunction with amino-acid residue.Fragment-NHCH (R ⁹)-B (OH) can be the R configuration.Yet the non-chiral centre that is limited to these conformations of the present invention.

In a compounds, P3 (S3-combination) amino acid and/or the amino acid whose side chain of P2 (S2-combination) are to be selected from the group of group shown in formula A or the B except hydrogen:

-(CO) _a-(CH ₂) _b-D _c-(CH ₂) _d-E (A)

-(CO) _a-(CH ₂) _b-D _c-C _e(E ¹)(E ²)(E ³) (B)

Wherein

A is 0 or 1;

E is 1;

B and d are 0 or integer independently, and (b+d) is 0-4 thus, perhaps

(b+e) be 1-4;

C is 0 or 1;

D is O or S;

E is H, C ₁-C ₆Alkyl, perhaps saturated or undersaturated cyclic group, it contains particularly 5-6 member's ring (for example phenyl) or 8-14 member's condensed ring system (for example naphthyl) of maximum 14 members usually, alkyl or cyclic group are optional to be replaced by being independently selected from following maximum three groups (for example group), and described group is C ₁-C ₆Trialkylsilkl ,-CN ,-R ¹³,-R ¹²OR ¹³,-R ¹²COR ¹³,-R ¹²CO ₂R ¹³With-R ¹²O ₂CR ¹³, R wherein ¹²Be-(CH ₂) _f-, R ¹³Be-(CH ₂) _gH, perhaps alkyl or cyclic group are optional is replaced by a kind of group, the non-hydrogen atom of this group is made up of heteroatoms in carbon atom and the ring, number is 5-14, it contains a loop systems (for example aryl) and an optional alkyl and/or alkylidene group, wherein f and g all are 0-10 independently, g is (although-OH also can be a substituting group) particularly at least 1, condition is that (f+g) is no more than 10, more particularly is no more than 6, the most particularly 1,2,3 or 4, and condition is that perhaps E is C if substituting group is to contain the described group of a loop systems then have only a replacement ₁-C ₆Trialkylsilkl; E ¹, E ²And E ³All be independently selected from-R ¹⁵With-J-R ¹⁵, wherein J is a 5-6 member's a ring, R ¹⁵Be selected from C ₁-C ₆Trialkylsilkl ,-CN ,-R ¹³,-R ¹²OR ¹³,-R ¹²COR ¹³,-R ¹²CO ₂R ¹³,-R ¹²O ₂CR ¹³And one or two kind of halogen (for example in one situation of back, be formation-J-R ¹⁵It is dichlorophenyl for a group), R wherein ¹²And R ¹³Be respectively as above-mentioned R ¹²Group and R ¹³Group is (at E ¹, E ²And E ³Contain R ¹³In the number acid of group, g is 0 or 1);

In formula (A) or in the group (B), any ring all is carbocyclic ring or aromatic ring, or the two; Replace by halogen especially F with arbitrary or a plurality of hydrogen atom of carbon atom bonding is all optional.

In certain embodiments, a is 0.If a is 1, c can be 0.In special embodiment, (a+b+c+d) He (a+b+c+e) be no more than 4, especially 1,2 or 3.(a+b+c+d) can be 0.

E, E ¹, E ²And E ³Group for example comprise aromatic ring such as phenyl, naphthyl, pyridyl, quinolyl (quinolinyl) and furyl; The unsaturated ring of non-aromatics, for example cyclohexenyl; Saturated ring such as cyclohexyl.E contains the two condensed ring system, for example a fluorenyl of aromatic ring and non-aromatic ring.One class E, E ¹, E ²And E ³Group is aromatic ring (comprising hetero-aromatic ring), especially 6 members' aromatic ring.In some compounds, E ¹Be H, while E ²And E ³Not H; In those compounds, E ²And E ³Group for example is phenyl (replacement or unsubstituted), C ₁-C ₄Alkyl, for example methyl.

In a class embodiment, it is C that E contains a replacement ₁-C ₆Alkyl, (C ₁-C ₅Alkyl) carbonyl, carboxyl C ₁-C ₅Alkyl, aryl (comprising heteroaryl), especially 5 members or preferred 6 members' aryl (for example phenyl or pyridyl), or aralkyl (for example arylmethyl or aryl ethyl, wherein aryl can be a heterocyclic, preferably 6 members).

In another kind of embodiment, it is OR that E contains a replacement ¹³, R wherein ¹³Can be 6 members' ring, it can be aromatic ring (a for example phenyl), and a replacement that perhaps contains is by this 6 members' cyclosubstituted alkyl (for example methyl or ethyl).

Group shown in one class formula A or the B be those wherein E be 6 members' aromatic ring, optional particularly at the 2nd or the 4th by-R ¹³Or-OR ¹³Replace.

The present invention includes such salt, wherein P3 and/or P2 side chain comprise wherein 1 or 2 hydrogen atom by halogen for example F or cyclic group of Cl metathetical.

The present invention includes such class salt, its Chinese style (A) or side chain (B) are as shown in the formula (C), (D) or (E):

Wherein q is 0-5, for example is 0,1 or 2, each T all be independently hydrogen, halogen (for example F or Cl) ,-SiMe ₃,-R ¹³,-OR ¹³,-COR ¹³,-CO ₂R ¹³Or-O ₂CR ¹³In structure (D) and some embodiments (E), T is the 4th at phenyl, and is-R ¹³,-OR ¹³,-COR ¹³,-CO ₂R ¹³Or-O ₂CR ¹³, and R ¹³Be C ₁-C ₁₀Alkyl, more particularly C ₁-C ₆Alkyl.In a subclass, T is-R ¹³Or-OR ¹³, for example wherein f and g all are 0,1,2 or 3 independently; In some side-chain radicals of this subclass, T is-R ¹²OR ¹³And R ¹³Be H.

In a class group, described side chain is suc as formula shown in (C), and each T all is R independently ¹³Or OR ¹³, and R ¹³Be C ₁-C ₄Alkyl.In some this compounds, R ¹³Be the ramose alkyl, it is a straight chain in other compound.In some groups, carbonatoms is 1-4.

In many two peptide fragment YCO-(this dipeptides can be the terminal protection of N-or not be), P3 amino acid has one as above-mentioned formula (A) or the side chain (B), and the P2 residue is an imino-acid.

Therefore the present invention comprises such medicine, and it comprises is thrombin inhibitors, particularly the salt of the organic boronic with neutral P1 (S1 in conjunction with) group of selective thrombin inhibitors metal-salt for example.About bind thrombin S3, the more information of the group in S2 and S1 site is seen for example Tapparelli C et al, Trends Pharmacol.Sci.14:366-376,1993; Sanderson Pet al, Current Medicinal Chemistry, 5:289-304,1998; Rewinkel J et al, Current Pharmaceutical Design, 5:1043-1075,1999; And Coburn C Exp.Opin.Ther.Patents 11 (5): 721-738,2001 is described.Zymoplasm inhibition salt of the present invention is non-be limited to describe in the publication of in last sentence, listing have S3, those of S2 and S1 affinity groups.

Described boric acid is about 100nM or lower to the Ki of zymoplasm, for example is about 20nM or lower.

A hypotype of acid comprises the acid shown in the formula (III) shown in the formula (I):

X is a group with N-terminal amino group bonding, can be that H is to form NH ₂The character of X is not crucial, but can be above-mentioned specific X group.In one embodiment, can be the benzyloxycarbonyl of mentioning.

In some instances, X is R ⁶-(CH ₂) _p-C (O)-, R ⁶-(CH ₂) _p-S (O) ₂-, R ⁶-(CH ₂) _p-NH-C (O)-or R ⁶-(CH ₂) _p-O-C (O)-, wherein p is 0,1,2,3,4,5 or 6 (preferred 0 and 1), R ⁶Be a H or 5-13 member's cyclic group, this group is optional by being selected from halogen, amino, nitro, hydroxyl, C ₅-C ₆Cyclic group, C ₁-C ₄Alkyl and the C that contains the interior oxygen of chain and/or be connected by oxygen in the chain with 5-13 member's cyclic group ₁-C ₄1,2 or 3 substituting group of alkyl replaces, and the aforesaid alkyl group is optional by being selected from halogen, amino, nitro, hydroxyl and C ₅-C ₆A substituting group of cyclic group replaces.More particularly, X is R ⁶-(CH ₂) _p-C (O)-or R ⁶-(CH ₂) _p-O-C (O)-, p is 0 or 1.A described 5-13 member's cyclic group is aromatic ring or hetero-aromatic ring normally, for example is 6 members' aromatic ring or hetero-aromatic ring group.In many cases, this group is not substituted.

The X group for example is (2-pyrazine) carbonyl, (2-pyrazine) alkylsulfonyl, particularly benzyloxycarbonyl.

Aa ¹Be the amino-acid residue with an alkyl side chain, this side chain contains and is no more than 20 carbon atoms (for example maximum 15 and optional maximum 13 carbon atoms) and comprises at least one cyclic group with maximum 13 carbon atoms.In certain embodiments, aa ¹Cyclic group 5 or 6 ring memberses are arranged.Aa for example ¹Cyclic group can be aryl, particularly phenyl.Typically, at aa ¹One or two cyclic group is arranged in the side chain.Some side chain comprises or by being formed by one or two 5 or 6 member's cyclosubstituted methyl.

More particularly, aa ¹Be Phe, Dpa or its be the hydrogenant analogue wholly or in part.The hydrogenant analogue is Cha and Dcha fully.

Aa ²It is an imino-acid residue that 4-6 ring members arranged.

One class product for example is to comprise wherein aa ²Be those products of the residue of imino-acid shown in the formula (IV):

R wherein ¹¹Be-CH ₂-, CH ₂-CH ₂-,-S-CH ₂-or-CH ₂-CH ₂-CH ₂-, when ring was 5 or 6 members, this group was chosen wantonly at one or more-CH ₂-group is by 1-3 C ₁-C ₃Alkyl replaces, for example to form R ¹¹Group-S-C (CH ₃) ₂-.For example these imino-acids are azetidine 2 carboxylic acids, particularly (s) azetidine 2 carboxylic acid, more especially proline(Pro).

From on institute as can be known, class product very preferably is by aa wherein ¹-aa ²Be those compositions of Phe-Pro.In another preferred classification, aa ¹-aa ²Be Dpa-Pro.In other product, aa ¹-aa ²Be Cha-Pro or Dcha-Pro.Certainly, comprise that also wherein Pro is by the corresponding product classification of (s)-azetidine 2 carboxylic acid metathetical.

R ⁹As described above, and can be formula-(CH ₂) _sRadicals R shown in the-Z ¹Integer s is 2,3 or 4, and W is-OH-OMe ,-OEt or halogen (F, Cl, I or preferred Br).Specify be the Z group be-OMe and-OEt, especially-OMe.In certain embodiments, be 3 for all Z group s, in fact all like this for all compounds of the present invention.Especially, R ¹Group is the 2-bromotrifluoromethane, 2-chloroethyl, 2-methoxy ethyl, 4-brombutyl, 4-chlorobutyl, 4-methoxyl group butyl, particularly 3-bromopropyl, 3-chloropropyl and 3-methoxy-propyl.More preferably, R ¹It is the 3-methoxy-propyl.The 2-ethoxyethyl group is another preferred R ¹Group.

Therefore, class salt very preferably is by formula X-Phe-Pro-Mpg-B (OH) ₂Especially formula Cbz-Phe-Pro-Mpg-B (OH) ₂Shown in those salt of acid form; Also preferably wherein Mpg have another particularly preferred R by one ¹The residue displacement of group and/or Phe are by Dpa or another aa ¹The analogue of these compounds of residue metathetical.

The aa of described salt ¹Group is the R configuration preferably.Aa ²Group is (S)-configuration preferably.Particularly preferred salt has the aa of (R)-configuration ¹And (S)-aa of configuration ²Chiral centre-NH-CH (R ¹)-B-is (R)-configuration preferably.Commercial formulation have (R, S, the R) chiral centre in arranging, for example in the situation of the salt of Cbz-Phe-Pro-BoroMpg-OH:

The present invention includes the salt of Cbz-(R)-Phe-(S)-Pro-(R)-boroMpg-OH (and formula X-(R)-Phe-(S)-Pro-(R)-boroMpg-OH shown in other compound), it is at least 90% pure, and for example at least 95% is pure.

In main term, salt of the present invention can be considered to corresponding to above-mentioned organic boronic and a kind of alkaline reaction product, for example alkali metal compound; Yet the non-product that derives from this reaction that is limited to of described salt, and can be the product that obtains by other approach.

Therefore described salt can obtain by acid shown in the formula (I) is contacted with a kind of highly basic.Therefore the present invention has contained the product (composition of matter) of the character with acid shown in the formula (I) and alkaline reaction product.Described alkali is that medicine is acceptable.

The suitable salt that can mention is:

1. metal-salt, particularly univalent metal, what can mention is basic metal;

2. the salt of strong basicity nitrogen-containing organic compound comprises:

2A. the salt of guanidine and analogue thereof;

2B. the salt of strong basicity amine for example comprises (i) aminosugar and (ii) other amine.

In above-mentioned salt, special instruction be basic metal, especially Na and Li.Aminosugar also has been described.

Specificity salt is the salt of acid boric acid (acid boronate), although in fact described acid salt can contain two deprotonation borates of considerably less part.Term " acid boric acid " is meant leg-of-mutton-B (OH) ₂Group, wherein one of B-OH group is deprotonation, and is meant corresponding tetrahedroid group in the balance.Acid boric acid has the stoichiometry consistent with single deprotonation.

Therefore the present invention comprises the product (composition of matter) that comprises the salt that can be represented by formula V:

Y wherein ^N+Be acceptable alkaline positively charged ion, the aa of deriving from of a kind of medicine ¹, aa ², X and R ¹As above-mentioned.The present invention also comprises wherein R ¹By another R ⁹The product of group displacement.

The solvability of one class salt is when determining in the dispersed system at 25mg/ml as described in embodiment, and its solvability is about 10mM or more, for example about at least 20mM.More particularly when determining in as the dispersed system at 50mg/ml as described in the embodiment, its solvability is 50mM at least.

The present invention comprises the salt of boric acid shown in the formula (I), and it observes stoichiometry and formula " (borate ^-) _nPositively charged ion ^N+" shown in the stoichiometry unanimity of salt.This salt of one class is expressed from the next:

[Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)(O ^-)]M ⁺

Wherein M+ represents univalent positively charged ion, especially an alkali metal cation.Should understand above-mentioned expression is the theoretic expression of product, and it is 1: 1 definitely that its observation stoichiometry may not reach fully.In following formula, the borate that trilateral is represented is always represented the borate of trilateral, tetrahedroid or blended trilateral/tetrahedroid.

The special such product of illustration, it comprises:

(i) be selected from (a) formula (VIII): X-(R)-Phe-(S)-Pro-(R)-Mpg-B (OH) ₂Shown acid, wherein X is H or amino protecting group, especially Cbz, reaches (b) kind of its borate anion; And

(ii) with the ion with valency n of described kind combination, described kind has and n with described ion: 1 theoretical kind: the observation stoichiometry that the ion chemistry metering is consistent.In a class salt, V is 1.

Consider counterion then:

1. monovalence metal, especially an alkali metal salt

Suitable basic metal comprises lithium, sodium and potassium.All these all are obviously soluble.Because its high dissolution illustration lithium and sodium.Lithium salts particularly sodium salt is remarkable high resolution with respect to sylvite.Use sodium in many situations.The salt that contains alkali metal mixture has been contained in the present invention.

The present invention includes the product of the salt that comprises formula (VI):

M wherein ⁺Be a kind of alkalimetal ion, aa ¹, aa ², X and R ¹As above-mentioned, the present invention also comprises such salt, and wherein two of the borate group oh groups all are that salt form (preferably has another identical M ⁺Group), reach the mixture of this salt.The present invention also comprises such product, wherein R ¹By another R ⁹Group displacement.

2. strong basicity nitrogen-containing organic compound

The present invention includes can be by the product (characteristic with the product that obtains by this reaction) as above-mentioned peptide boric acid and a kind of strong organic bases reaction acquisition.The preferred organic bases of two classes is described among 2A and the 2B hereinafter.Particularly preferably be acid salt (wherein one of two boric acid-OH groups are deprotonations).The most common ground, described salt contains the organic balanced ion (not considering contaminant trace species) of single type, yet the present invention also relates to contain the salt of organic balanced ion mixture; In a subclass, described different counterion all belongs to following paragraph 2A family, perhaps also might belong to following paragraph 2B family; At another subclass, it not is the organic balanced ionic mixture that all comes from same family (2A or 2B) that described salt comprises.

Suitable organic bases comprise pKb be 7 or higher, for example 7.5 or those higher organic basess, as 8 or higher scope.The preferred lower alkali [at least one polar functional group (for example 1,2 or 3 this group) for example being arranged ,] of lipotropy as hydroxyl; Therefore aminosugar is the preferred alkali of a class.

2A. guanidine and analogue thereof

Guanidine compound (guanidine) can be that any soluble and medicine can be accepted compound in principle, and it has the guanidino group of a guanidine radicals or replacement, and perhaps one replaces or unsubstituted guanidine analogue.Suitable substituents comprises aryl (for example phenyl), and alkyl or the alkyl that is interrupted by an ehter bond or thioether bond in any situation, typically contain 1-6,1,2,3 or 4 carbon atom especially, as in the situation of methyl or ethyl.Described guanidino group can have 1,2,3 or 4 substituted radicals, but 1 or 2 substituted radical is arranged usually, for example endways on the nitrogen.One class guanidine is single hydrocarbylation; Another kind of is dialkylization.What can mention as the guanidine analogue is sulphur guanidine and 2-aminopyridine.Have unsubstituted guanidino group, for example guanidine and special category of arginic compound formation.

The present invention relates to contain the salt of guanidine mixture.

A kind of special guanidine compound is L-arginine or L-arginine analog, for example D-arginine, the perhaps D-or the preferred L-isomer of homoarginine or smart agmatine [(the amino butyl of 4-) guanidine].Not too preferred arginine analog for example is the NG-nitro-L-arginine methyl esters, and the guanidine analogue, particularly 2-aminopyrimidine of restriction, for example 2, and 6-quinazoline diamines is as 5,6,7,8-tetrahydrochysene-2,6-quinazoline diamines.The guanidine radicals mixture also can be a kind of peptide, for example contains arginic dipeptides; Such dipeptides is L-tyrosyl-L-arginine.

Some special guanidine compounds are the compounds shown in the formula (VII):

Wherein n formula 1-6 for example is 2 at least, for example 3 or more, be no more than 5 in many cases.The most especially, n is 3,4 or 5.R ²Be H or carboxylate salt or deutero-carboxylate salt, for example to form a kind of ester (C for example ₁-C ₄Alkyl ester) or acid amides.R ³Be H, C ₁-C ₄The residue of the natural or alpha-non-natural amino acid (for example tyrosine) of alkyl or.Compound shown in the formula (IV) is the L-configuration normally.Compound is arginine (n=3 shown in the formula (IV); R ²=carboxyl; R ³=H) and arginine derivative or analogue.

The present invention includes the product of the salt that comprises formula (IX):

Aa wherein ¹, aa ², X and R ¹As described above, G ⁺Be the acceptable organic compound of a kind of medicine of protonated form, it comprises a guanidino group or its analogue, the present invention also comprise two oh groups of borate group wherein all be salt form (preferably have another identical G ⁺Group) the salt and the mixture of this salt.The present invention also comprises wherein R ¹By another R ⁹The product of group displacement.

2B. strong basicity amine

The present invention includes can be by the product (characteristic with the product that obtains by this reaction) of above-mentioned peptide boric acid and a kind of highly basic reaction acquisition, and described highly basic is a kind of amine.Described amine can be any soluble and acceptable amine of medicine in principle.

Therefore the amine of one suitable class comprises those that also have polar functional group except single amine groups, and this compound more wetting ability is stronger and compare more solvable with other compound.In some salt, functional group described or that each is extra all is hydroxyls.Some amine have 1,2,3,4,5 or 6 extra functional groups, especially oh groups.For example in a class amine, (amino adds oh group): the ratio of carbon atom is 1: 2 to 1: 1, preferred especially back one ratio.These amine with one or more extra polar functional group can be hydrocarbon, especially the alkane that is replaced by amino group and extra polar group.Described amino group can be replacement or unsubstituted, and gets rid of amino the replacement, and polarity alkali for example can comprise 10 carbon atoms at the most; Usually be no less than 3 this carbon atoms, for example 4,5 or 6.Aminosugar is included in the category of this polarity alkali.

The present invention includes the product of the salt that comprises formula (X):

Aa wherein ¹, aa ², X and R ¹As described above, A ⁺Be the acceptable amine of a kind of medicine of protonated form, the present invention comprises that also wherein two hydroxyls of borate group all are that salt form (preferably has another identical A ⁺Group) the salt and the mixture of this salt.In this product of a class, A ⁺Be a kind of amine as the described protonated form of paragraph 2B (i); In another classification, A ⁺Be as (ii) a kind of amine of described protonated form of 2B.The present invention also comprises wherein R ¹By another R ⁹The product of group displacement.

Paragraph 2B (i) and 2B (ii) for example understand two class amine alkali.Particularly preferably be acid salt (wherein one of two boric acid-OH groups are deprotonations).The most common ground, described salt contains the amine counterion (not considering contaminant trace species) of single type, yet the present invention also relates to contain the salt of amine counterion mixture; In a subclass, described different counterion all belongs to following paragraph 2B (i) family, perhaps also might belong to (ii) family of following paragraph 2B; At another subclass, it not is the organic balanced ionic mixture that all belongs to same family (2B (i) or 2B are (ii)) that described salt comprises.

2B (i) aminosugar

The characteristic of aminosugar is not crucial to the present invention.Preferred aminosugar comprises sugar, the especially glycosamine of open loop.Also preferred annular aminosugar.One class aminosugar is that N-terminal is unsubstituted, and another kind of preferably N-terminal is replaced by one or two (for example one) N-terminal substituting group.Suitable substituents for example is a hydrocarbyl group, and non-being limited to contained 1-12 carbon atom; Described substituting group can comprise alkyl or aryl group or these two.Substituting group for example is C ₁, C ₂, C ₃, C ₄, C ₅, C ₆, C ₇And C ₈Alkyl group, particularly methyl and ethyl, illustration methyl.Data show aminosugar, especially N-methyl D-glycosamine has very high solvability.

A kind of most preferred aminosugar is N-methyl D-glycosamine:

2B is other amine (ii)

Other suitable amine comprises amino acid whether (no matter natural generation), and its side chain is by an amino group, especially Methionin replaces.

Some amine are the compounds shown in the formula (XI):

N wherein, R ²And R ³With identical shown in the formula (IV).The compound of formula (VI) is the L-configuration normally.The compound of formula (VI) is Methionin (n=4; R ²=carboxyl; R ³=H) and lysine derivative or analogue.Most preferred amine is L-Methionin.

Other suitable amine is nitrogen heterocyclic ring.At least common this heterogeneous ring compound is alicyclic; The heterocycle compound is that N replaces, and another kind of preferably N is unsubstituted.Described heterocycle can contain 6 rings and form atom, as in the situation of piperidines, piperazine and morpholine.One class amine comprise by polar substituent, especially hydroxyl replace contain the N heterocycle, for example replace 1,2 or 3 time.

Therefore the present invention comprises the amine except aminosugar, and it also has one or more (for example 1,2,3,4,5 or 6) polar substituent, especially hydroxyl except an amine groups.(amino adds oh group) of this compound: the ratio of carbon atom is 1: 2-1: 1, and preferred especially back one ratio.

The present invention includes mixed salt, promptly contain the salt of the mixture of boric acid peptide group and/or counterion, but preferred single salt.

The salt of solid form can contain a kind of solvent, for example water.The present invention includes wherein said salt is an anhydrous class product basically.The present invention comprises that also wherein said salt is a class product of hydrate.

The application of product of the present invention

Salt of the present invention is thrombin inhibitors.Therefore it be used for Trombin inhibiting.Therefore the invention provides the compound that has control extravasated blood and especially suppress the agglutinative potentiality, for example the secondary incident after treatment or prevention myocardial infarction form.The medical use of described compound can be preventative (comprise the treatment thrombosis and prevent thrombotic generation) and curative (comprising that the prevention thrombosis takes place once more or secondary thrombus forms incident).

When needs antiprothrombin preparation, can use described salt.In addition, have been found that described salt comprises those salt of the boric acid shown in the formula (III), can be used for the treatment of artery thrombosis by treatment or prevention.Therefore the salt that is disclosed illustrates can treat or prevent animal to comprise thrombosis and hypercoagulability in people's blood and the tissue.Term " thrombosis " comprises that for example marantic clot formation, artery thrombosis, intracardial thrombus formation, Coronary thrombosis, crawl row thrombosis, infective thrombus formation, mesentery thrombosis, placenta thrombosis, propagated thrombus form, traumatic thrombus forms and venous thrombosis.

Known hypercoagulability can cause thrombotic disease.

Can for example comprise vein obstruction with the venous thromboembolism of compounds for treating of the present invention or prevention, subpulmonic obstruction (pulmonary infarction), dvt forms, the thrombosis relevant with cancer and cancer chemotherapy, be secondary to disease such as lack of protein c with thrombophilia, Protein S lacks, the thrombosis of Antithrombin III shortage and factor V Leiden, and derive from the thrombosis of the disease such as the systemic lupus erythematous (struvite connective tissue disease) of acquired thrombophilia.For venous thromboembolism, compound of the present invention is used to keep the opening of inlying catheter.

Can for example comprise thromboembolic stroke (thrombus that separates that causes the neuroscience pathology relevant), the heart source property thromboembolism relevant with the heart source property thromboembolism of compounds for treating of the present invention or prevention with atrial fibrillation (atrium, erratic top myokymia fast) with cerebral blood supply insufficiency, relevant heart source property thromboembolism, and the heart source property thromboembolism of being correlated with the heart valve of repairing such as heart valve prosthesis with heart disease.

The illness that comprises artery thrombosis for example comprises unstable angina (chest comes from serious squeezing pain coronarius), myocardial infarction (because blood is for not enough cardiomyocyte cell death that causes), ischemic heart disease (because blood is for blocking local asphyxia due to (as stricture of artery)), during the percutaneous transluminal coronary angioplasty or obturation inaccessible again, restenosis, coronary artery bypass graft behind percutaneous transluminal coronary angioplasty afterwards, and obstructive cerebrovascular disease.With regard to arteriovenous (mixed) thrombosis, antithrombotic of the present invention forms the opening that compound also is used to keep the arteriovenous branch road.

Other illness with hypercoagulability and thrombotic disease blood vessel that can mention is secondary to or obtains the shortage, homocysteine mass formed by blood stasis of shortage, circulation anti-phospholipid antibody (lupus anti-coagulant) from Antithrombin II, heparin-induced thrombocytopenia and fibrinolysis defective.

The special purpose that can mention comprises therapeutic and/or preventative processing venous thrombosis and pulmonary infarction.The preferable use of product of the present invention (the particularly salt of TRI 50c) comprising:

Prevention venous thromboembolism (for example dvt forms and/or pulmonary infarction).The patient who for example comprises experience plastic surgery operations such as whole hip replacement, whole knee prosthesis, main hip or knee surgery; Be in the patient of experience common surgical procedures in the thrombosis high-risk state, as the belly or the pelvic surgery of cancer; Reach the patient who lies in bed more than 3 days and have acute heart failure, acute respiratory failure, infection.

Thrombosis among prevention blood samples of patients dialyzing access, the end-stage renal disease patient.

No matter whether prevention end-stage renal disease patient's central vessel pathology (death, myocardial infarction etc.) need hemodialysis.

Patient's medium sized vein thromboembolic events of chemotherapy is accepted in prevention by inlying catheter.

Thromboembolic events among the patient of prevention experience artery of lower extremity reconstruction operations (bypass, end-artery surgical blanking, puncture vessel plasty etc.).

Treatment venous thromboembolism incident.

With the cardiovascular event (for example unstable angina, no Q ripple myocardial ischemia/infraction) in the another kind of cardiovascular treatment agent combination prevention of acute coronary syndromes, described another kind of cardiovascular treatment agent for example is an acetylsalicylic acid (acetylsalicylic acid; Acetylsalicylic acid is the trade mark of having registered in Germany), thrombolytic agent (example sees below), anti-platelet agent (example sees below).

Suffers from the patient of Acute Myocardial Infarction with acetylsalicylic acid, thrombolytic agent (example sees below) combined therapy.

Therefore thrombin inhibitors of the present invention can be used for aforementioned all illnesss for the treatment of and/or preventing property processing.

In a method, product of the present invention is used for the treatment of the patient of hemodialysis, by product of the present invention is provided in dialysis solution, as in WO 00/41715 about as described in other thrombin inhibitors.Therefore the present invention comprises dialysis solution and comprises the dialysate concentrate of a kind of product of the present invention, and the present invention also comprises the method for the dialysis patients of this treatment of treatment needs, and described method comprises uses a kind of dialysis solution that comprises low molecular weight thrombin inhibitor.The present invention also comprises the application of antithrombotic product of the present invention in the medicine of production for treating dialysis patients, and antithrombotic wherein of the present invention forms product and provides in dialysis solution.

In another method, product of the present invention is used to resist disadvantageous cell proliferation, as in WO 01/41796 about as described in other thrombin inhibitors.Described disadvantageous cell proliferation is typical disadvantageous hyperplasia sexual cell propagation, for example smooth muscle cell, the propagation of vascular smooth muscle cell especially.Find that product of the present invention is particularly useful in treatment inner membrance hyperplasia, the part of vascellum endometrial hyperplasia is a smooth muscle cell proliferation.Restenosis can be considered to because due to the new intima hyperplasia; Therefore the inner membrance hyperplasia comprises restenosis in the specification sheets of the present invention.

Product of the present invention is also expected can treat ischemic disease.More particularly, they can be used for the ischemic disease that treatment (no matter therapeutic or preventative) suffers from or be in the patient in non-valve atrial fibrillation (NVAF) danger, as among the WO 02/36157 about as described in other thrombin inhibitors.Ischemic disease is the illness that its result comprises the restriction of body part blood flow.This term is understood to include thrombosis and the hypercoagulability in blood, tissue and/or the organ.The special purpose that can mention comprises and prevents and/or treats ischemic heart disease, myocardial infarction, systemic embolism for example in kidney or spleen, reach more especially cerebral ischemia, comprise cerebral thrombosis, cerebral embolism and/or the cerebral ischemia relevant (in other words with non-cerebral thrombosis or embolism, i.e. treatment (no matter therapeutic still is preventative) thrombotic or ishemic stroke and treatment transient ischemic attacks), particularly treatment suffers from or is in patient in the NVAF danger.

Product of the present invention is also expected can treat rheumatic/arthritis disease, as in WO03/007984 about as described in other thrombin inhibitors.Therefore, product of the present invention can be used for treating chronic arthritis, rheumatoid arthritis, osteoarthritis or ankylosing spondylitis.

In addition, expect that product of the present invention is used to prevent behind thrombolysis, puncture shaping surgery (PTA) and the crown arterial bypass inaccessible (being thrombosis) again; Prevent after general microsurgery and the vascular surgery thrombosis once more.The purposes of product of the present invention further comprises the disseminated inravascular coagulation that the treatment for the treatment of and/or preventing property is caused by bacterium, many places wound, poisoning or any other mechanism; When contacting, the valve of external source surface as blood vessel graft, intravascular stent, vessel catheter, mechanicalness and biological reparation in blood and the body or any other medical apparatus carry out the anticoagulation processing; And, blood carries out the anticoagulation processing as during making motive-lung machine carry out operation on vessels of heart or in hemodialysis the time when contacting with medical apparatus outside the body.

The purposes of product of the present invention comprises that further wherein there is disadvantageous excessive zymoplasm in treatment and does not have the illness of hypercoagulability sign, for example neurodegenerative disease such as alzheimer's disease.Except the effect of zymoplasm to the aggegation process, known its can activate a large amount of cells (as neutrophilic granulocyte, inoblast, endotheliocyte and smooth muscle cell).Therefore, compound of the present invention can be used for also that the treatment for the treatment of and/or preventing property is special sends out property and adult respiratory distress syndrome, pulmonary fibrosis, septic shock, septicemia, inflammatory reaction after radiotherapy or chemotherapy are handled, comprise but the non-oedema that is limited to acute or CAS such as coronary artery disease, cerebral arterial disease, peripheral arterial disease, reperfusion injury and restenosis after percutaneous puncture plasty (PTA).

Described salt can also be used for the treatment of pancreatitis.

Salt of the present invention further can be used for suppressing the thrombocyte procoagulant activity.The invention provides a kind of method that suppresses the thrombocyte procoagulant activity, by suffering from or being in Mammals, the particularly salt of a kind of boric acid of the present invention of people in the artery thrombosis danger.The present invention also provides the application of this salt in producing the medicine that suppresses the thrombocyte procoagulant activity.

Based on such observation, boric acid promptly of the present invention shows effective inhibition artery thrombosis and venous thrombosis to product of the present invention as the application of thrombocyte procoagulant activity inhibitor.

The illness that comprises artery thrombosis comprises acute coronary syndrome (especially myocardial infarction and unstable angina), cerebrovascular thrombosis and peripheral arterial are inaccessible to be reached owing to atrial fibrillation, valvular heart disease, arteriovenous shut, the artery thrombosis that inlying catheter or coronary stent take place.Therefore, the method that the present invention provides a kind of treatment to be selected from these diseases or illness on the other hand comprises giving especially a kind of salt of the present invention of human patients of Mammals.The present invention includes and be used for for example product of coronary stent or other artery implant of artery environment, it has a bag quilt that comprises salt of the present invention.

Salt preventability of the present invention be used for the treatment of be sure of to be in suffer from artery thrombosis or comprise the disease of artery thrombosis or the danger of illness in individuality, perhaps therapeutic is used (comprise prevention is thrombotic take place once more or the thrombosis of secondary).

Therefore the present invention includes the application of described selective thrombin inhibitors (organic borate) in preventative or the above-mentioned disease of therapeutic treatment, with and application in the production of pharmaceutical preparation and pharmaceutical preparation.

Administration and pharmaceutical preparation

Described salt can give the host, for example has in the active situation of antiprothrombin at described medicine, to obtain the antiprothrombin effect.In the situation than large animal such as people, described compound can give separately or give with medicine acceptable diluent, vehicle or carrier combinations.Term " medicine is acceptable " comprises that people and animal doctor's purpose are all acceptable, wherein preferred human medicinal application acceptability.In case of oral administration, described compound is preferably to prevent that the form that salt of the present invention contacts with acidic gastric juice from giving, and the casing dosage form prevents that like this salt of the present invention from discharging until reaching duodenum in this way.

Casing for example is suitably to be made by carbohydrate polymer or polyethylene polymer.The casing raw material for example comprises but the non-rhodia phthalic ester that is limited to, the rhodia succinate, Mierocrystalline cellulose hydrogen phthalic ester, the rhodia trimellitate, ethyl cellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethyl rhodia succinate, carboxymethylethylcellulose, starch acetate phthalic ester, amylose starch acetate phthalic ester, polyvinyl acetate phthalic ester, poly-vinyl butyrate phthalic ester, the vinylbenzene acid/maleic acid copolymers, methacrylic acid-Sipacril 2739OF (MPM-05), methacrylic acid-methacrylic acid-methylmethacrylate copolymer (MPM-06), and methyl vinylformic acid-Sipacril 2739OF (Eudragit

L﹠amp; S)..Optional described casing contains a kind of softening agent.Softening agent for example comprises but the non-triethyl citrate that is limited to, triactin and diethyl phthalate.

Salt of the present invention can make up and/or give jointly with any cardiovascular treatment agent.Commercial applications, clinical evaluation and clinical before a large amount of available cardiovascular treatment agent are arranged in the research and development, can be selected with product of the present invention by the medicinal composition treatment with preventing cardiovascular disease.This preparation can be to be selected from but non-one or more preparation that is limited to several primary categories, and promptly fat-reducing medicament comprises IBAT (ileum Na ⁺/ cholic acid cotransporter) inhibitor, the special class medicine (fibrate) of shellfish, nicotinic acid, statin, CETP (cholesteryl ester transfer protein) inhibitor, and cholic acid chelating agent, antioxidant, comprise vitamin-E and probucol (probucol), the IIb/IIIa antagonist (abciximab for example, eptifibatide, tirofiban), aldosterone inhibitor (spironolactone (spirolactone) and epoxymexrenone) for example, adenosine A 2 receptor antagonists (for example losartan), adenosine A 3 receptor agonists, beta-Blocking agent, acetylsalicylic acid, loop diuretic and ACE (Zinc metallopeptidase Zace1) inhibitor.

Salt of the present invention can make up and/or give jointly with any antithrombotic agent with different mechanism of action, as the anti-platelet agent acetylsalicylic acid, ticlopidine, clopidogrel, thromboxane acceptor and/or synthetase inhibitors, prostacyclin mimetics and phosphodiesterase inhibitor and ADP-acceptor (P ₂T) antagonist.

Product of the present invention is being treated thrombotic disease, can further made up and/or give jointly with thrombolytics in myocardial infarction especially, described thrombolytics such as tissue plasminogen activator (natural, reorganization or modification), streptokinase, urokinase, uPA, methoxybenzoyl Profibrinolysin-streptokinase activator mixture (APSAC), animal sialisterium plasminogen activator or the like.

Salt of the present invention can make up and/or for example adenosine A 1 or A3 receptor stimulant give jointly with a kind of cardioprotective agent.

The present invention also provides the treatment patient a kind of method of inflammatory disease, comprise with product of the present invention with NSAID for example cox 2 inhibitor treat this patient.This disease comprises but the non-ephritis that is limited to, systemic lupus erythematous, rheumatoid arthritis, glomerulonephritis, vasculitis and sarcoidosis.Therefore, antithrombotic formation salt of the present invention can make up and/or give jointly with NSAID.

Therefore, typically, salt of the present invention can give the host to obtain a kind of zymoplasm restraining effect, any other zymoplasm inhibition or anti-thrombosis function of perhaps mentioning among the present invention.

The actual dose level of activeconstituents can change in the pharmaceutical composition of the present invention, to obtain effectively to reach at particular patient, composition and mode of administration the amount (the present invention is referred to as " treatment significant quantity ") of the active compound that required treatment replys.Selected dosage level depends on the activity of specific compound, the seriousness of the illness of being treated and the patient's who is treated state and present illness history.Yet the initial dose of the known described compound of those skilled in the art is lower than the needed level of the therapeutic action that reaches hope, progressively increases dosage then until the effect that reaches hope.

For example, be expected at present in the situation of salt of orally give TRI 50c, described salt for example can every day gives the amount of 0.5-2.5mg/Kg for twice, and meter is made the amount of TRI 50c.Other salt can give by equimolar amount.The present invention gives non-being limited to this quantity or scheme, dosage and the scheme of sentence outside described before being included in.

According to a further aspect in the invention, provide a kind of oral drug preparation, it comprises product of the present invention and mixes the acceptable adjuvant of a kind of medicine, diluent or carrier.

The solid dosage of oral administration comprises capsule, tablet (being also referred to as pill), powder and particle.In this solid dosage, described active compound typically mixes at least a inert pharmaceutically-acceptable excipients or carrier such as Trisodium Citrate or Lin Suanergai and/or one or more following raw material: a) filling agent or extender such as starch, lactose, sucrose, glucose, N.F,USP MANNITOL and silicic acid; B) tackiness agent such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and Sudan Gum-arabic (acacia); C) wetting agent such as glycerine; D) disintegrating agent such as agar-agar, lime carbonate, potato or tapioca (flour), alginic acid, some silicate and yellow soda ash; E) solution retarding agent such as paraffin; F) absorption enhancer such as quaternary ammonium compound; G) wetting agent such as hexadecanol and Zerol; H) absorption agent such as kaolin and bentonite and i) lubricant such as talcum, calcium stearate, Magnesium Stearate, solid polyethylene glycol, sodium lauryl sulphate and composition thereof.In the situation of capsule and tablet, described formulation can also comprise buffer reagent.The solids composition that also can use similar type in the gelatine capsule of soft hard filling for example uses lactose and high-molecular weight polyoxyethylene glycol as vehicle as filling agent.

Suitably, oral preparations can contain a kind of dissolving subsidiary.Described dissolving subsidiary non-being limited to is its character, as long as it is that medicine is acceptable.For example comprise nonionogenic tenside, as sucrose fatty ester, glycerol fatty acid ester, sorbitan fatty(acid)ester (for example Witconol AL 69-66), polyoxyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, Voranol EP 2001, methoxyl group Polyethoxyolefin alkyl oxide, polyoxyethylene alkyl phenyl ether, cithrol, polyoxyethylene alkyl amine, polyoxyethylene alkyl thioether, polyoxyalkylene polyoxypropylene multipolymer, polyoxyethylene glycerol fatty acid ester, pentaerythritol fatty ester, the propylene glycol mono fatty acid ester, polyoxyethylene propylene glycol mono fatty acid ester, polyoxyethylene sorbitol fatty acid ester, fatty acid alkyl alcohol amide, and alkyl amine oxide; Bile acide and salt thereof (gallodesoxycholic acid for example, cholic acid, Septochol, Felacrinos and salt thereof, and glycine or its taurine conjugate); Ionic surface active agent, as sodium lauryl sulphate, fatty acid soaps, alkylsulfonate, alkylphosphonic, phosphoric acid ether, the soap of basic aminoacids; Trolamine soap, and alkyl quaternary ammonium salts; And amphoterics such as trimethyl-glycine and aminocarboxylate.

Described active compound also can be the microencapsulation form, if suitably then have one or more above-mentioned vehicle.

The liquid dosage form of oral preparations comprises the acceptable milk sap of medicine, solution, suspension, syrup and elixir.Except active compound, this liquid dosage form can contain this area inert diluent such as water or other solvent commonly used, solubilizing agent and emulsifying agent such as ethanol, Virahol, ethyl-carbonate, ethyl acetate, phenylcarbinol, peruscabin, propylene glycol, 1,3 butylene glycol, dimethyl formamide, oil (particularly cotton oil, peanut oil, Semen Maydis oil, germ oil, sweet oil, Viscotrol C and sesame oil), glycerine, tetrahydrofurfuryl alcohol, fatty acid ester of polyoxyethylene glycol and sorbitan and composition thereof.Except inert diluent, oral compositions can also comprise adjuvant such as wetting agent, emulsifying agent and suspension agent, sweeting agent, seasonings and spices.Suspension also can contain suspension agent such as ethoxylation isooctadecanol except described active compound, polyoxyethylene sorbitol and sorbitan ester, and Microcrystalline Cellulose, an aluminium hydroxide, bentonite, agar-agar, and tragacanth gum and composition thereof.

Product of the present invention can be a granulous solid form in small, broken bits, for example can be micronized.Powder or grain in small, broken bits can incapsulate.

Described activated mixture can single dose, multiple dosage or give as sustained release preparation.

Should understand the pharmaceutical product that the invention provides the dried fine particulate form that is suitable for administration from aforementioned content, it comprises the metal-salt of boric acid shown in the formula (I), for example an alkali metal salt.Described metal-salt can be to be suitable for oral form.Preferably a kind of acid salt of described metal-salt.

Synthetic

1. peptide/peptide mimics is synthetic

The boric acid peptide comprises that for example the synthetic of Cbz-D-Phe-Pro-BoroMpg-O tetramethyl ethylene ketone is well known to those skilled in the art, and described in the prior art field, comprise Claeson et al (US 5574014 and other) and Kakkar et al (WO 92/07869 and patent families comprise US5648338).Elgendy et al Adv.Exp.Med.Biol. (USA) 340:173-178,1993; Claeson, G.et al Biochem.J.290:309-312,1993; Deadman et al J.Enzyme Inhibition 9:29-41,1995 and Deadman et al J.Med.Chem.38:1511-1522,1995 to this also in addition description.

Can use definite methodology to carry out (Elgendy et al Tetrahedron.Lett.33:4209-4212,1992 in that the stereoselectivity of chirality B end carbon S or R configuration is synthetic; WO92/07869 and patent families comprise US 5648338), use (+) or (-)-pinane glycol as the directed thing (chiral director) of chirality (Matteson et al J.Am.Chem.Soc.108:810-819,1986; Matteson et al Organometallics.3:1284-1288,1984).To be the indispensable aminoboronic acid ester intermediate (for example Mpg-BO tetramethyl ethylene ketone) of dissolving obtain (the R)-isomer of wishing and with itself and dipeptides group (Cbz-(R)-Phe-(S)-Pro for example with selectivity to another kind method, identical with Cbz-D-Phe-L-Pro) coupling, this will form the residuum of described molecule.

Described boric acid peptide can the boric acid ester form synthesize at first, particularly has the ester of glycol.As mentioned below, this diol ester can change peptide boric acid into.

2. ester is to the conversion of acid

Can be with a kind of peptide boric acid ester such as Cbz-(R)-Phe-Pro-BoroMpg-O tetramethyl ethylene ketone hydrolysis to form corresponding acid.

A kind of new technology that changes the diol ester of peptide boric acid shown in the formula (I) into acid comprises this diol ester is dissolved in ether especially in the dialkyl ether, thus dissolved glycol and a kind of glycol amine for example dioxane hydramine reaction form precipitated product, reclaim this precipitation, be dissolved in it in polar organic solvent and dissolved product and for example aqueous acid reaction of a kind of aqueous medium thus, form peptide boric acid.Described boric acid can reclaim from the organic layer that gets self-reacting mixture, is for example undertaken by removing to desolvate, and desolvates as removing by vaporising under vacuum or distillation.Reaction between diol ester and the glycol amine can be carried out under for example refluxing.

The character of glycol is not crucial.The suitable glycol that can mention is aliphatics and aromatic substance, its have adjacent carbons or on the carbon atom that is replaced by another carbon substituted oh group.That is to say that suitable glycol comprises having in the chain or the compound of at least two oh groups that separated by at least two continuous carbon atoms in the ring.One class glycol comprises the hydrocarbon that is accurately replaced by two oh groups.This glycol be a kind of be tetramethyl ethylene ketone, another kind is the pinane glycol; What can also mention is neopentyl glycol, 1,1,2-propylene glycol, 1, ammediol, 2,3-butyleneglycol, 1,2-di-isopropyl ethylene glycol, 5,6-decanediol and 1,2-dicyclohexyl ethylene glycol.

The alkyl group of dialkyl ether preferably has 1,2,3 or 4 carbon atoms, and described these alkyl groups can be identical or different.A kind of ether of illustrative is diethyl ether.

The alkyl group of dioxane hydramine preferably has 1,2,3 or 4 carbon atoms, and described these alkyl groups can be identical or different.A kind of dioxane hydramine of illustrative is a diethanolamine.Diethanolamine/acid reaction product is in room temperature hydrolysis in water, and hydrolysis rate can quicken by adding acid or alkali.

Polar organic solvent is CHCl preferably ₃Other example is generally many alkyl halides and ethyl acetate.Any polar organic solvent except alcohol all is acceptable in principle.

Aqueous acid is 1 strong inorganic acid suitably for pH, example hydrochloric acid.

Behind acid-respons, reaction mixture is used for example NH suitably ₄Cl or the washing of another kind of weak base.

For example the following stated of specific procedure:

1. tetramethyl ethylene ketone or the pinane diol ester with selected peptide boric acid is dissolved in the diethyl ether.

2. add diethanolamine and with mixture 40 ℃ of backflows.

3. remove (filtration) precipitated product, with diethyl ether or the another kind of polar organic solvent washing (usually several times) except alcohol, and dry (for example by vacuum-evaporation).

4. desciccate is dissolved in a kind of organic solvent CHCl for example except alcohol ₃In, adding acid or alkali aqueous solution, hydrochloric acid (pH1) for example was in stirring at room mixture for example 1 hour.

5. take out organic layer and use NH ₄The Cl solution washing.

6. evaporate organic solvent and dry remaining solid product.

Aforesaid method causes forming " the glycol amine affixture " of the peptide boric acid that can be called formula (I) easily, particularly has this affixture of diethanolamine, the present invention includes this affixture.The molecular structure of this affixture is also unknown, and they may comprise a kind of compound, in this compound two of glycol amine oxygen and nitrogen all with the boron coordination; They can comprise ion.Yet described affixture is considered to ester.But a kind of specific new product that the present invention comprises is compound, particularly (R shown in the through type VIII, S, R)-product that the tetramethyl ethylene ketone of TRI 50c or the reaction of pinane diol ester and diethanolamine obtain, be that new product is (R, S, R)-TRI 50c/ diethanolamine " affixture ", wherein said acid is (R, S, R)-TRI 50c.

Glycol amine of the present invention can be a kind of composition that comprises following material:

(i) material shown in the formula (XII)

Wherein X is H or an amino protecting group, the boron atom optional extra with the nitrogen-atoms coordination, the valency situation of terminal oxygen be opening (they can invest on another covalent linkage, and ion turns to-O ^-, some other for example intermediate state is perhaps arranged); And be in the bonding.

(ii) material shown in the formula (XIII)

Wherein the valency state of nitrogen-atoms and two Sauerstoffatoms is open.Should recognize that the terminal Sauerstoffatom of kind shown in the formula (IX) and the Sauerstoffatom of kind shown in the formula (X) can be identical Sauerstoffatoms, the kind of the kind of formula (X) and formula (IX) forms a kind of diol ester in this case.

Be understood that aforementioned techniques comprises a kind of method that reclaims the organic boronic product, described method comprises provides a kind of mixture that is dissolved in the solvent, this mixture comprises the organic boronic of soluble form and has the compound (being glycol amine) of two oh groups and an amino group, make the reaction of described organic boronic and glycol amine form precipitation, reclaim described precipitation.The organic boronic of soluble form can be aforesaid a kind of diol ester.Described solvent can be aforesaid a kind of ether.Described organic boronic can be one of organic boronic of mentioning in this specification sheets, and for example it can be formula (I), (II) or the organic boronic (III).Method described in this section is new and has formed one aspect of the present invention.Recovery method is to filter.

Reaction between the organic boronic of glycol amine and soluble form is suitably carried out at elevated temperatures, for example carries out under refluxing.

Another aspect of the present invention is the method that reclaims organoboron compound, and described method comprises:

Be provided in a kind of organic boronic of soluble form in the ether, for example a kind of medicine is suc as formula the compound shown in (III);

Be formed at a kind of solution of the soluble form in the ether;

With described solution and the combination of a kind of dioxane hydramine, make the organic boronic reaction of dioxane hydramine and soluble form form a kind of insoluble precipitation; And

Reclaim described precipitation.

Term in the last paragraph " solvable " be meant reaction medium than precipitated product in more soluble basically compound.In the changing method of this method, described ether is replaced by toluene or another kind of aromatic solvent.

Above-mentioned diethanolamine precipitation technology for example is another kind of novel method, and this is a kind of method of the tetramethyl ethylene ketone or the pinane diol ester of recovering peptide boric acid from ethereal solution, comprises diethanolamine is dissolved in the solution, makes precipitation form and reclaim this precipitation.The changing method of this method has been contained in the present invention, wherein uses the another kind of glycol except tetramethyl ethylene ketone or pinane glycol.

Described throw out, i.e. " affixture " can be changed into free organic boronic, for example undertaken by it is contacted with acid.Described acid can be a kind of aqueous acid, for example above-mentioned a kind of aqueous inorganic acid.Described precipitation can for example be dissolved in the organic solvent, contacts with described acid more afterwards.

Therefore the present invention provides a kind of method of producing organic boronic, comprises and changes its glycol amine reaction product into acid.

The acid that derives from preceding two sections described methods can be changed into the salt of this acid and a kind of polyvalent metal, and this salt is formulated into a kind of pharmaceutical composition of oral dosage form subsequently.

3. salt is synthetic

Normally, described salt can be by contacting the salt of wishing to form with corresponding peptide boric acid with a kind of suitable highly basic.In the situation of metal-salt, the oxyhydroxide of metal is suitable alkali (perhaps for example can use the carbonate of metal), sometimes be that described acid is contacted with a kind of corresponding metal alkoxide (for example methylate) more expediently, corresponding for this reason alkanol is a kind of suitable solvent.The salt of organic bases can prepare by peptide boric acid and organic bases are contacted self.Described salt for example is acid salt (one-BOH proton is replaced), produces acid salt with a kind of monovalent cation, and described bronsted lowry acids and bases bronsted lowry suitably reacts with basic equimolar amount.Therefore the acid of convention: the alkali molar ratio is n substantially: 1, and wherein n is the cationic valency of alkali.

In a program, with peptide boric acid in can with the miscible organic solvent of the water solution in acetonitrile or the alcohol (for example ethanol, methyl alcohol, propyl alcohol Virahol for example, or another kind of alkanol) for example, with the aqueous solution combination of alkali.Make the bronsted lowry acids and bases bronsted lowry reaction and reclaim salt.This reaction is typically carried out in envrionment temperature (for example 15-30 ℃, for example 15-25 ℃), but can use the temperature of rising, for example until the boiling point of reaction mixture, but more commonly lower temperature, and for example at the most 40 ℃ or 50 ℃.Standing and reacting mixture or stirring (normally stirring).

The reaction times of bronsted lowry acids and bases bronsted lowry is not crucial, but what found to wish is to keep reaction mixture at least one hour.The reaction times of one or two hour is normally suitable, can certainly adopt the longer reaction times.

Salt can reclaim as evaporating or precipitating through any suitable method from reaction mixture.Precipitation can be undertaken by adding excessive miscible solvent, and described salt solubleness in this miscible solvent is limited.In a preferred technology, salt is to reclaim by reaction mixture is vacuumized drying.Preferably with the salt purifying, for example, for example be evacuated to drying and purifying afterwards by filtering gained solution after salt is dissolved again and being dried.Again the dissolving can make water for example distilled water carry out.Then salt is further purified, for example removing remainder water, this can be by at suitable solvent, and further dissolving again among ethyl acetate or the THF is evaporated to drying subsequently and carries out.Purifying procedure can carry out (being 15-30 ℃, for example 15-25 ℃) in envrionment temperature, perhaps for example is no more than 40 ℃ or 50 ℃ in the temperature that suitably raises and carries out; For example salt can be dissolved in water and/or the solvent by stirring, heats or does not heat to for example 37 ℃.

The present invention also comprises the method for a kind of drying salt of the present invention and other peptide boric acid salt, and described method comprises they are dissolved in organic solvent for example among ethyl acetate or the THF, for example is evaporated to drying by vacuumizing then.

The preferred solvent that is generally used for purification of salts is ethyl acetate or THF, perhaps perhaps is another kind of organic solvent.

The general procedure of the salt of synthetic Cbz-Phe-Pro-BoroMpg-OH is as follows:

With Cbz-Phe-Pro-BoroMpg-OH (20.00g, 38.1mM) room temperature through stirring and dissolving in acetonitrile (200ml).In this solution, add the necessary alkaline solution in distilled water (190ml); The alkali that adds is as the 0.2M solution of monovalent cation.Make the gained clear soln for example by leaving standstill or stirring reaction for some time, in arbitrary situation, be 1-2 hour.Typically react (for example 15-30 ℃, for example 15-25 ℃) in room temperature, but or can elevated temperature (for example being at most 30 ℃, 40 ℃ or 50 ℃).Then reaction mixture is evacuated to drying under vacuum, temperature is no more than 37 ℃, typically produces the frangible solid of a kind of white or a kind of oily/mucus.This oily/mucus is dissolved in the minimum distilled water (200ml-4L) again typically heat (for example to 30-40 ℃), at the most 2 hours usually.By filter paper filtering solution and be evacuated to drying, solution temperature also is no more than 37 ℃, perhaps freeze-drying.Products therefrom under vacuum dried overnight with the easy crushed solid of a kind of white of normal generation.If product exists with oily or viscous liquid state, then be dissolved in it in ethyl acetate then and be evacuated to drying, produce white solid.This white solid typical case is a kind of coarse amorphousness powder.

In the variant of aforementioned general procedure, acetonitrile is replaced with the mixable organic solvent of water by another kind of, and described solvent is particularly as above-mentioned alcohol, particularly ethanol, methyl alcohol, Virahol or another kind of propyl alcohol.

In the situation that solubility is lower in the salt formation reaction medium that a kind of organic borate is being selected, be not easy to directly from corresponding bronsted lowry acids and bases bronsted lowry, prepare, so the lower salt of solubility can be from preparing in more soluble a kind of salt this reaction medium.

The present invention also provides the application of boric acid in producing salt of the present invention.The present invention also comprises a kind of method for preparing product of the present invention, comprise for example formula (I), (II) or the boric acid (III) contact with the alkali that can produce this salt.

Be used to prepare that the peptide boric acid typical case has GLP or GMP character shown in the formula (I) of pharmaceutical preparation, perhaps meet GLP (" medicine non-clinical study quality control procedure ") or GMP (" Good Manufacturing Practice and Quality Control of Drug "; The present invention includes this acid.

Similarly, described acid is normally aseptic and/or be suitable for that pharmacology uses, and an aspect of of the present present invention relates to a kind of composition, and it is aseptic or is suitable for that pharmacology is used or comprises these two, and comprises the peptide boric acid shown in the formula (I).This composition can be particulate form or liquor or dispersion.

Intermediate acid can be unpack format, the present invention includes this isolating acid, particularly suc as formula the isolating acid of peptide boric acid shown in (VIII):

X-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH) ₂ (VIII)

Wherein X is that H is (to form NH ₂) or an amino protecting group.

It is as microparticle compositions that a kind of typical way of intermediate acid is provided, and said composition mainly is made up of this peptide boric acid, the present invention includes these compositions.Peptide boric acid accounts at least 75% of said composition weight usually, is typically to account at least 85% of said composition weight, for example accounts at least 95% of composition weight.

The another kind of typical way that intermediate acid is provided is as a kind of liquid composition, and said composition is by the dissolving of peptide boric acid shown in the formula (II) and peptide boric acid or be suspended in wherein a kind of liquid vehicle and form or dissolved by peptide boric acid shown in the formula (II) and peptide boric acid substantially or a kind of liquid vehicle of being suspended in is wherein formed.Described liquid vehicle can be an aqueous medium, water for example, or alcohol is as methyl alcohol, ethanol, Virahol or another kind of propyl alcohol, the mixture of another kind of alkanol or aforementioned medium.

The composition of intermediate acid is normally aseptic.Said composition can contain the peptide boric acid of particle shape formula in small, broken bits so that further processing.

The separation of steric isomer

The steric isomer of peptide boric acid ester or synthetic intermediate aminoboronic acid ester can split by for example any known way.Especially, the steric isomer of boric acid ester can split by HPLC.

Embodiment

Embodiment 1 to 3 and embodiment 33-introduction

Instrument:

In the program of following embodiment 1-3 and embodiment 33, use the standard laboratory glassware, and the professional utensil of operation and transfer air susceptibility reagent.

All glass waress are before use all 140-160 ℃ of heating at least 4 hours, then in moisture eliminator or by concentrating heat and using and do nitrogen gas stream blowing cooling.

Solvent:

The organic solvent that uses among embodiment 1-3 and the embodiment 33 all is anhydrous.Suitably, with they process sodium wire dryings before using.

Dry:

In the drying program of embodiment 1-3 and embodiment 33, test the degree of drying (drying that comprises organic solvent) of product by observation minimizing of weight in drying process.Minimizing according to weight in the following program determination drying process: with sample place vacuum drier and 40 ℃ at 100mbar dry 2 hours.When in drying the minimizing of weight be lower than the initiator gross weight 0.5% the time, think that product is an exsiccant.

Embodiment 1-3 has described the execution of following reaction scheme and the process that gained TRI 50c changes its sodium salt:

The LDA=LDA

LiHMDS=hexamethyldisilazane lithium is also referred to as two (trimethyl silyl) acid amides lithiums

Embodiment 1-TRI 50D's is synthetic

Step 1:Z-DIPIN B

Program A

Add 17.8g (732.5mmole) magnesium Xuan bits, 0.1g (0.4mmole) iodine and 127ml anhydrous tetrahydro furan also are heated to backflow.66g (608mmole) the 1-chloro-3-methoxy propane that adds 15ml subsequently in the 185ml anhydrous tetrahydro furan solution and under refluxing, stir until kickback takes place.After initial heat release stops, slowly adding 1-chloro-3-methoxy propane solution and be consumed until whole magnesium to keep gentle reflux.After reaction is finished, reaction mixture is cooled to envrionment temperature, and the solution of (620mmole) trimethyl-boron hydrochlorate in the 95ml anhydrous tetrahydro furan that slowly adds 64.4g; Back one solution is cooled to below 0 ℃, if it heats up during reaction process, then reaction mixture must add very lentamente with the temperature that keeps this solution and is lower than 65 ℃.After adding is finished, make reaction mixture be warming up to about 0 ℃ and restir 60 minutes.Slowly add the solution of 22.4ml sulfuric acid in 400ml water then, be lower than 20 ℃ to keep temperature.This layer is avaled and separate each phase.Aqueous layer is washed 3 times with the 200ml t-butyl methyl ether again.The organic layer of combination is avaled and remove isolated extra water from this solution.Organic layer is passed through dried over mgso and is evaporated to dried.Residue on evaporation filters from precipitated solid and will filter thing and is dissolved in the 175ml toluene.34.8g (292mmole) tetramethyl ethylene ketone is added in this solution, be no less than 10 hours in the envrionment temperature stirring subsequently.This solution evaporation to doing, is dissolved in the 475ml normal heptane and with the saturated sodium bicarbonate aqueous solution of 290ml and washs 3 times.N-heptane solution is evaporated to dry doubling reclaims fraction at 0.1-0.5mbar with the residue on evaporation distillation and at Bp 40-50 ℃.

Boiling point: 40-50 ℃/0.1-0.5mbar

Yield: 40.9g (70%) Z-DIPIN B (oil)

Program B

Add 17.8g (732.5mmole) magnesium Xuan bits, 0.1g (0.4mmole) iodine and 127ml anhydrous tetrahydro furan also are heated to backflow.66g (608mmole) the 1-chloro-3-methoxy propane that adds 15ml subsequently in the 185ml anhydrous tetrahydro furan solution and under refluxing, stir until kickback takes place.After initial heat release stops, slowly adding 1-chloro-3-methoxy propane solution to keep gentle reflux.After reaction is finished, reaction mixture is cooled to envrionment temperature, and the solution of (620mmole) trimethyl-boron hydrochlorate in the 95ml anhydrous tetrahydro furan that slowly adds 64.4g, the temperature of this solution is lower than-65 ℃.After adding is finished, make reaction mixture be warming up to about 0 ℃ and restir 60 minutes.Slowly add the solution of 22.4ml sulfuric acid in 400ml water then, be lower than 20 ℃ to keep temperature.Organic solvent is removed by distilling under vacuum.The 300ml normal heptane is added in the aqueous solution of residue on evaporation, add 34.8g (292mmole) tetramethyl ethylene ketone subsequently.Two-phase mixture is no less than 2 hours in the envrionment temperature stirring.After this layer is avaled, separate the water-based phase.The 300ml normal heptane is added water-based be no less than 2 hours mutually and at envrionment temperature stirring two-phase mixture.After this layer is avaled, separate the water-based phase.The combination organic layer is also used the 200ml water washing once, washs once with the saturated sodium hydrogen carbonate solution of 200ml subsequently, uses twice of 200ml water washing again.N-heptane solution is evaporated to dry doubling reclaims fraction at 0.1-0.5mbar with the residue on evaporation distillation and at Bp 40-50 ℃.

Boiling point: 40-50 ℃/0.1-0.5mbar

Yield: 40.9g (70-85%) Z-DIPIN B (oil)

Step 2:Z-DIPIN C

Add 16.6g (164mmole) Diisopropylamine and 220ml tetrahydrofuran (THF) and be cooled to-30 to-40 ℃.In this solution, add 41.8g (163mmole) n-Butyl Lithium solution of 25% in normal heptane, stirred 1 hour at 0 to-5 ℃ subsequently.The diisopropyl amide lithium solution of this prepared fresh is cooled to-30 ℃, between-60 and-75 ℃, adds solution and 35.5g (418mmole) methylene dichloride of 27.9g (139mmole) Z-DIPIN B in the 120ml tetrahydrofuran (THF) then.In this temperature stirred solution half an hour, be incorporated in 480ml (240mmole) 0.5N Zinc Chloride Anhydrous (II) or 32.5g (240mmole) anhydrous solid zinc chloride (II) in the tetrahydrofuran (THF) subsequently.After 1 hour, make reaction mixture be warming up to envrionment temperature-65 ℃ of stirrings, restir 16-18 hour.Reaction mixture is evaporated to dried (promptly desolvating until removing), adds the 385ml normal heptane subsequently.Reaction mixture washs with 150ml 5% sulfuric acid, 190ml saturated sodium bicarbonate solution and 180ml saturated nacl aqueous solution.Organic layer filters and is evaporated to dried (promptly desolvating until removing) through dried over mgso.The oiliness resistates need not to be further purified and carries out next procedure.

Yield: 19g (55%) Z-DIPIN C

Step 3:Z-DIPIN D

In the solution of 400ml tetrahydrofuran (THF), add 25% solution of 34.7g (136mmole) n-Butyl Lithium in normal heptane to 23.8g (148mmole) hexamethyldisilazane, and stirred 1 hour at-15 ℃.This solution is cooled to-55 ℃, adds 30.6g (123mmole) the Z-DIPIN C that is dissolved in the 290ml tetrahydrofuran (THF) subsequently, and in the LiHMDS of this prepared fresh solution, add the 35ml tetrahydrofuran (THF).Make solution be warming up to envrionment temperature and stirred 12 hours.Reaction mixture is evaporated to dried, residue on evaporation is dissolved in the 174ml normal heptane, with 170ml water and the saturated sodium chloride solution washing of 75ml.Dried fully (promptly desolvating until removing) filtered and be evaporated to organic phase through dried over mgso.The oiliness resistates is dissolved in the 100g normal heptane.This solution need not to be further purified and carries out next step.

Yield: 32.2g (70%) Z-DIPIN D

Step 4:Z-DIPIN (TRI50b, rough)

26.6g (71mmole) solution of Z-DIPIN D in the 82.6g normal heptane dilutes with the 60ml normal heptane and is cooled to-60 ℃, imports 10.5g (285mmole) hydrogenchloride subsequently.Reaction mixture vacuumizes subsequently and discharges with nitrogen, with about 20 ℃ increasing degree temperature is risen to envrionment temperature simultaneously.From oily precipitation, remove and desolvate and with the fresh normal heptane replacing of 60ml several times.The oily resistates is dissolved in the 60ml tetrahydrofuran (THF) (solution A).

Add the 130ml tetrahydrofuran (THF) in another flask, 24.5g (61.5mmole) Z-D-Phe-Pro-OH and 6.22g (61.5mmole) N-methylmorpholine also are cooled to-20 ℃.In this solution, add 8.4g (61.5mmole) isobutyl chlorocarbonate in the 20ml tetrahydrofuran (THF) solution and stirred 30 minutes, add solution A at-25 ℃ subsequently.When adding finishes, be added to many 16ml (115mmole) triethylamine to regulate pH to 9-10, the pH value is measured with the pH test paper.Still under nitrogen, make reaction mixture be warming up to envrionment temperature and stirred 3 hours.Solvent evaporated also is dissolved in evaporation residue in the 340ml t-butyl methyl ether (t-BME).Twice of 175ml1.5% salt pickling of the solution of Z-DIPIN in t-BME.The acidic cleaning liquid that merges is washed 1 time with 175ml t-BME again.The organic layer that merges washs with 175ml water, 175ml saturated sodium bicarbonate solution, 175ml 25% sodium chloride solution, and drying is also filtered on sal epsom.This solution need not to be further purified and carries out next step.

Yield: 29.9g (80%) Z-DIPIN

Embodiment 2-TRI 50D's (the diethanolamine affixture of TRI 50C) is synthetic

The initiator that uses among this embodiment is the TRI 50b solution (Z-DIPIN) that obtains in embodiment 1.This solution need not to be further purified and directly carries out synthesizing of TRI 50d.Z-DIPIN solution that will be in t-BME (contains 7.0g (11.5mmole) (R) TRI50b calculates based on the HPLC result of Z-DIPIN for R, S) and is evaporated to driedly, and residue on evaporation is dissolved in the 80ml diethyl ether.Add 1.51g (14.4mmole) diethanolamine and with mixture in reflux at least 10 hours, product precipitation during this period.Suspension is cooled to 5-10 ℃, filters filter residue with the diethyl ether washing.

Be improvement chirality and chemical purity, wet filter residue (7g) is dissolved in the 7ml methylene dichloride, be cooled to 0-5 ℃, by adding 42ml diethyl ether precipitated product and filtering.With isolating wet product 35 ℃ in vacuum-drying, perhaps at least 4 hours until 1 day.

Yield: 5.5g (80%) Tri50d

Fusing point: 140-145 ℃

The preparation of the sodium salt of embodiment 3-TRI50C

1.5kg (2.5mole) TRI50d that will obtain in embodiment 2 is dissolved in the 10.5L methylene dichloride.Add 11L 2% hydrochloric acid and with mixture in stirring at room 30 minutes at the most (optional about 20 minutes).In organic phase, form precipitation.After stirring, this layer is avaled and separate.With aqueous layer 2.2L methylene dichloride washed twice again.The organic layer of the combination solution washing of 625g ammonium chloride in 2.25L water.(pH of chloride buffer aqueous extract at about pH 1-2 to about pH4-5 scope) because strong acidic condition can the cracking peptide bond.Organic phase is passed through dried over mgso, filters and will filter thing to be evaporated to dried.The amount that acid is changed into required solvent of salt and alkali is calculated in the boric acid analysis (the RP HPLC method by embodiment 28 is carried out 30 minutes (optional about 20 minutes) at the most in room temperature) that dissociates.If obtain the free acid of 2.5mol, then residue on evaporation is dissolved in the 5L acetonitrile, add 5% solution of 100g (2.5mole) sodium hydroxide in 2.2L water subsequently.Solution was stirred 2 hours in envrionment temperature (for example 15-30 ℃, optional room temperature), evaporate in vacuum (ca.10mmHg) in the temperature that is no more than 35 ℃ then.Be dissolved in residue on evaporation in the fresh acetonitrile of 3.5L again and be evaporated to and do to remove the water of trace.If residue on evaporation is done, then it is dissolved in the 3L acetonitrile (perhaps 6L THF), slowly add in the mixture of 32L normal heptane and 32L diethyl ether.Enough add lentamente to avoid product to form bulk or viscous, the joining day is no less than 30 minutes.Filter out precipitated product, with the normal heptane washing and in vacuum-drying, temperature is initially about 10 ℃, is increased to about 35 ℃ of limits then until exsiccation.

Yield: 1.0kg (70%) Tri50c sodium salt.

The program of embodiment 1-3 can be amplified in proportion, if careful operation can produce high-purity salt.In the diethanolamine settling step, importantly (R) TRI 50b uses 1.25 normal diethanolamine for R, S whenever amount.In the hydrolysis of diethanolamine ester, importantly avoided contacting with aqueous acid for a long time.Equally, TRI 50b should be synthetic by the Grignard reaction of Z-DIPIN A.

The another kind of embodiment 4-TRI 50B to TRI 50C changes

The nitrogen that the synthesis program of describing in this and subsequently synthetic embodiment normally is being purchased and using carries out under the dried solvent.

1. the TRI 50b of about 300g that will obtain by the HPLC purifying of racemic TRI 50b is dissolved in about 2.5L diethyl ether.The isomer purity of the TRI 50b of estimation different batches is at 85%R, and S, R extremely surpass 95%R, and S is in the R scope.

2. add about 54ml diethanolamine (with 1: 1 stoichiometry of total TRI 50b content), and with mixture 40 ℃ of backflows.

3. remove precipitated product, with diethyl ether washing several times and dry.

4. the dry labor thing is dissolved in CHCl ₃In.Add hydrochloric acid (pH 1) and with mixture about 1 hour in stirring at room.

5. remove organic layer and use NH ₄The Cl solution washing.

6. organic solvent and dry remaining solid product are removed in distillation.

Typical yields: about 230g

The preparation of the lithium salts of embodiment 5-TRI50C

(20.00g 38.1mM) is dissolved under stirring at room in the acetonitrile (200ml) Cbz-Phe-Pro-BoroMpg-OH that will obtain by the method for embodiment 4.In this solution, add LiOH 0.2M solution of (190ml) in distilled water.The gained settled solution stirring at room 2 hours, is vacuumized drying then under vacuum, temperature is no more than 37 ℃.Gained oil/mucus is dissolved in the 500ml distilled water again, must be with heating slightly about 20 minutes.Through filter paper filtering and be evaporated to driedly, solution temperature is no more than 37 ℃ again with solution.Products therefrom spends the night to the easy crushed solid of a kind of white of normal acquisition in vacuum-drying.

Then salt is dried to constant weight (72 hours) in vacuum through silica.

Yield 17.89g.

Trace analysis:

C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)	Metal % observed value (calculated value)
C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)	Metal % observed value (calculated value)	57.14 (61.03)	6.60 (6.64)	7.34 (7.90)	2.07 (2.03)	Li 1.26 (1.31)

The UV/ visible spectrum of the lithium salts of embodiment 6-TRI50C

The salt of program that record derives from embodiment 5 in distilled water at 20 ℃ at the UV/ of 190nm-400nm visible spectrum.The given λ of salt _Max210 and 258nm.The weight of measuring the salt of doing then is to calculate optical extinction coefficient.Use the λ of 258nm _MaxUse following formula to calculate optical extinction coefficient:

A=ε cl wherein A is an absorbancy

C is a concentration

L is the latus rectum length in UV pond

ε is an optical extinction coefficient.

Optical extinction coefficient: 451

The water solubility of the lithium salts of embodiment 7-TRI50C

The salt that uses among this embodiment is to use embodiment 5 described method corrects to make.The difference of the method for being revised is the TRI 50c of 100mg as initiator, and the product that is dissolved in the water again is dry by freeze-drying, and filters by 0.2 μ m filter membrane.Be sure of that salt contains about 85% R, S, R isomer.

Be to determine maximum water solubility, the dried salt of 25mg shaken in water at 37 ℃ that filtered sample is also measured UV spectrum.This salt remains a kind of undissolved white residue.Lithium salts is more soluble, therefore is dissolved into 50mg/ml again in previous described same mode.

When solubleness when 25mg/ml dissolves is 43mM (23mg/ml),

When solubleness when 50mg/ml dissolves is 81mM (43mg/ml).

The preparation of the sodium salt of embodiment 8-TRI50C (TGN 255)

(20.00g 38.1mM) is dissolved under stirring at room in the acetonitrile (200ml) Cbz-Phe-Pro-BoroMpg-OH that will obtain by the method for embodiment 4.In this solution, add the 0.2M solution of NaOH in distilled water (190ml).Stirring at room 2 hours, vacuumize down dryly then the gained settled solution, temperature is no more than 37 ℃.Gained oil/mucus is dissolved in the 500ml distilled water again, and heated slightly about 15-20 minute.With filter paper filtering solution and be evaporated to driedly, solution temperature is no more than 37 ℃ again.Products therefrom under vacuum dried overnight to the easy crushed solid of a kind of white of normal harvest.This product is dissolved in it in ethyl acetate in this case and is evaporated to and do to produce the white solid product owing to the existence of surplus water is oily or mucus shape.

Then salt is dried to constant weight (72 hours) through silica under vacuum.

Yield: surpass 50%.

Trace analysis:

C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)	Metal % observed value (calculated value)
C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)	Metal % observed value (calculated value)	59.93 (59.24)	6.47 (6.44)	7.31 (7.67)	1.91 (1.98)	Na 3.81 (4.20)

The UV/ visible spectrum of the sodium salt of embodiment 9-TRI50C

The sodium salt of program that record derives from embodiment 8 in distilled water at 20 ℃ at the UV/ of 190nm-400nm visible spectrum.The given λ of salt _Max210 and 258nm.The weight of measuring the salt of doing then is to calculate optical extinction coefficient.Use the λ of 258nm _MaxUse following formula to calculate optical extinction coefficient:

A=ε cl wherein A is an absorbancy

C is a concentration

L is the latus rectum length in UV pond

ε is an optical extinction coefficient.

Optical extinction coefficient: 415

The water solubility of the sodium salt of embodiment 10-TRI50C

The salt that uses among this embodiment is to use embodiment 8 described method corrects to make.The difference of the method for being revised is the TRI 50c of 100mg as initiator, and the product that is dissolved in the water again is dry by freeze-drying, and filters by 0.2 μ m filter membrane.Be sure of that salt contains about 85% R, S, R isomer.

Be to determine maximum water solubility, the dried salt of 25mg shaken in water at 37 ℃ that filtered sample is also measured UV spectrum.This salt remains a kind of undissolved white residue.Sodium salt is more soluble, therefore is dissolved into 50mg/ml again in previous described same mode.

When solubleness when 25mg/ml dissolves is 44mM (25mg/ml),

When solubleness when 50mg/ml dissolves is 90mM (50mg/ml)

The preparation of the sylvite of embodiment 11-TRI50C

(20.00g 38.1mM) is dissolved under stirring at room in the acetonitrile (200ml) Cbz-Phe-Pro-BoroMpg-OH that will obtain by the method for embodiment 4.In this solution, add the 0.2M solution (190ml) of KOH in distilled water.The gained settled solution stirring at room 2 hours, is vacuumized drying then under vacuum, temperature is no more than 37 ℃.Gained oil/mucus is dissolved in the 1L distilled water again, heat to 37 ℃ about 2 hours.With filter paper filtering solution and be evaporated to driedly, solution temperature is no more than 37 ℃ again.Products therefrom under vacuum dried overnight with the easy crushed solid of a kind of white of normal harvest.

Yield: 14.45mg.

Then salt is dried to constant weight (72 hours) through silica under vacuum.

Trace analysis:

C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)	Metal % observed value (calculated value)
C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)	Metal % observed value (calculated value)	54.84 (57.55)	6.25 (6.26)	7.02 (7.45)	2.01 (1.92)	K4.29 (6.94)

The UV/ visible spectrum of the sylvite of embodiment 12-TRI50C

The sylvite of program that record derives from embodiment 11 in distilled water at 20 ℃ at the UV/ of 190nm-400nm visible spectrum.The given λ of TRI50C and salt _Max210 and 258nm.The weight of measuring the salt of doing then is to calculate optical extinction coefficient.Use the λ of 258nm _MaxUse following formula to calculate optical extinction coefficient:

A=ε cl wherein A is an absorbancy

C is a concentration

L is the latus rectum length in UV pond

ε is an optical extinction coefficient

Optical extinction coefficient: 438.

The water solubility of the sylvite of embodiment 13-TRI50C

The salt that uses among this embodiment is to use embodiment 11 described method corrects to make.The difference of the method for being revised is the TRI 50c of 100mg as initiator, and the product that is dissolved in the water again is dry by freeze-drying, and filters by 0.2 μ m filter membrane.Be sure of that salt contains about 85% R, S, R isomer.

Be to determine maximum water solubility, the dried salt of 25mg shaken in water at 37 ℃ that filtered sample is also measured UV spectrum.This salt remains a kind of undissolved white residue.

When solubleness when 25mg/ml dissolves is 29mM (16mg/ml)

The preparation of the zinc salt of embodiment 14-TRI 50C

The relative solubility of zinc hydroxide is such, if this oxyhydroxide uses the program of embodiment 5 to be used to prepare corresponding TRI 50c salt, they can not cause the salt formation of homogeneity.Therefore need to disclose a kind of new method with the preparation zinc salt, as reaching at this described in embodiment hereinafter.

(2.24g 4.10mM) is dissolved in the distilled water (100ml) in room temperature TRI 50c sodium salt, carefully is incorporated in zinc chloride (4.27ml, 0.5M) also stirring among the THF.Leach the white precipitate that forms immediately and use distilled water wash.This solid is dissolved in the ethyl acetate also with distilled water wash (2 * 50ml).Organic solution is vacuumized drying and the white solid that produces was passed through silica dry 3 days in moisture eliminator, carry out trace analysis afterwards.Yield 1.20g.

¹HNMR 400MHz，δ _H(CD ₃OD)7.23-7.33(20H，m，ArH)，5.14(4H，m，PhCH ₂O)，4.52(4H，m，αCH)，3.65(2H，m)，3.31(12H，m)，3.23(6H，s，OCH ₃)，2.96(4H，d，J 7.8Hz)，2.78(2H，m)，2.58(2H，m)，1.86(6H，m)，1.40(10H，m)。

¹³C NMR 75MHz δ _C(CD ₃OD)178.50，159.00，138.05，137.66，130.54，129.62，129.50，129.07，128.79，128.22，73.90，67.90，58.64，58.18，56.02，38.81，30.06，28.57，28.36，25.29。

FTIR(KBr disc)v _max(cm ^-1)3291.1，3062.7，3031.1，2932.9，2875.7，2346.0，1956.2，1711.8，1647.6，1536.0，1498.2，1452.1，1392.4，1343.1，1253.8，1116.8，1084.3，1027.7，916.0，887.6，748.6，699.4，595.5，506.5。

The preparation of the arginic acid salt of embodiment 15-TRI50C

(20.00g 38.1mM) is dissolved under stirring at room in the acetonitrile (200ml) Cbz-Phe-Pro-BoroMpg-OH that will obtain by the method for embodiment 4.In this solution, add the 0.2M solution of arginine in distilled water (190ml).The gained settled solution stirring at room 2 hours, is vacuumized drying then under vacuum, temperature is no more than 37 ℃.Gained oil/mucus is dissolved in the 2L distilled water again, heat to 37 ℃ about 2 hours.With filter paper filtering solution and vacuumize drying, solution temperature is no more than 37 ℃ again.Products therefrom under vacuum dried overnight with the easy crushed solid of a kind of white of normal harvest.

Then gained salt is dried to constant weight (72 hours) through silica under vacuum.

Yield: 10.54g.

Trace analysis:

C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)
C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)	52.47 (56.65)	7.12 (7.20)	15.25 (14.01)	1.52 (1.54)

The UV/ visible spectrum of the arginic acid salt of embodiment 16-TRI50C

The salt of program that record derives from embodiment 15 in distilled water at 20 ℃ at the UV/ of 190nm-400nm visible spectrum.The given λ of TRI50C and salt _Max210 and 258nm.The weight of measuring the salt of doing then is to calculate optical extinction coefficient.Use the λ of 258nm _MaxUse following formula to calculate optical extinction coefficient:

A=ε cl wherein A is an absorbancy

C is a concentration

L is the latus rectum length in UV pond

ε is an optical extinction coefficient

Optical extinction coefficient: 406.

The water solubility of the arginic acid salt of embodiment 17-TRI50C

The salt that uses among this embodiment is to use embodiment 15 described method corrects to make.The difference of the method for being revised is the TRI 50c of 100mg as initiator, and the product that is dissolved in the water again is dry by freeze-drying, and filters by 0.2 μ m filter membrane.Be sure of that salt contains about 85% R, S, R isomer.

When solubleness when 25mg/ml dissolves is 14mM (10mg/ml).

The preparation of the lysine salt of embodiment 18-TRI50C

(20.00g 38.1mM) is dissolved under stirring at room in the acetonitrile (200ml) Cbz-Phe-Pro-BoroMpg-OH that will obtain by the method for embodiment 4.In this solution, add the 0.2M solution of Methionin in distilled water (190ml).The gained settled solution stirring at room 2 hours, is vacuumized drying then under vacuum, temperature is no more than 37 ℃.Gained oil/mucus is dissolved in the 3L distilled water again, heat to 37 ℃ about 2 hours.With filter paper filtering solution and vacuumize drying, solution temperature is no more than 37 ℃ again.Products therefrom under vacuum dried overnight with the easy crushed solid of a kind of white of normal harvest.This product can be oily or mucus shape (because due to remaining water), is dissolved in product in the ethyl acetate in this case and vacuumizes dry to produce white solid product.

Then gained salt is dried to constant weight (72 hours) through silica under vacuum

Yield: 17.89.

Trace analysis:

C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)
C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)	57.03 (59.11)	7.43 (7.36)	10.50 (10.44)	1.72 (1.61)

The UV/ visible spectrum of the lysine salt of embodiment 19-TRI50C

The salt of program that record derives from embodiment 18 in distilled water at 20 ℃ at the UV/ of 190nm-400nm visible spectrum.The given λ of TRI50C and salt _Max210 and 258nm.The weight of measuring the salt of doing then is to calculate optical extinction coefficient.Use the λ of 258nm _MaxUse following formula to calculate optical extinction coefficient:

A=ε cl wherein A is an absorbancy

C is a concentration

L is the latus rectum length in UV pond

ε is an optical extinction coefficient

Optical extinction coefficient: 437.

The water solubility of the lysine salt of embodiment 20-TRI50C

The salt that uses among this embodiment is to use embodiment 18 described method corrects to make.The difference of the method for being revised is the TRI 50c of 100mg as initiator, and the product that is dissolved in the water again is dry by freeze-drying, and filters by 0.2 μ m filter membrane.Be sure of that salt contains about 85% R, S, R isomer.

When solubleness when 25mg/ml dissolves is 13mM (8.6mg/ml).

The preparation of the N-methyl-D-glucamine salt of embodiment 21-TRI50C

(20.00g 38.1mM) is dissolved under stirring at room in the acetonitrile (200ml) Cbz-Phe-Pro-BoroMpg-OH that will obtain by the method for embodiment 4.In this solution, add the 0.2M solution of N-methyl D-glycosamine in distilled water (190ml).The gained settled solution stirring at room 2 hours, is vacuumized drying then under vacuum, temperature is no more than 37 ℃.Gained oil/mucus is dissolved in the 500ml distilled water again, and heated slightly about 20 minutes.With filter paper filtering solution and be evaporated to driedly, solution temperature is no more than 37 ℃ again, or freeze-drying.Products therefrom under vacuum dried overnight with the easy crushed solid of a kind of white of normal harvest.

Yield: 21.31g.

Trace analysis:

C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)
C% observed value (calculated value)	H% observed value (calculated value)	N% observed value (calculated value)	B% observed value (calculated value)	56.67 (56.67)	7.28 (7.41)	7.74 (7.77)	1.63 (1.50)

The UV/ visible spectrum of the N-methyl-D-glucamine salt of embodiment 22-OF TRI50C

The salt of program that record derives from embodiment 21 in distilled water at 20 ℃ at the UV/ of 190nm-400nm visible spectrum.The given λ of TRI50C and salt _Max210 and 258nm.The weight of measuring the salt of doing then is to calculate optical extinction coefficient.Use the λ of 258nm _MaxUse following formula to calculate optical extinction coefficient:

A=ε cl wherein A is an absorbancy

C is a concentration

L is the latus rectum length in UV pond

ε is an optical extinction coefficient

Optical extinction coefficient: 433.

The water solubility of the N-methyl-D-glucamine salt of embodiment 23-TRI50C

The salt that uses among this embodiment is to use embodiment 21 described method corrects to make.The difference of the method for being revised is the TRI 50c of 100mg as initiator, and the product that is dissolved in the water again is dry by freeze-drying, and filters by 0.2 μ m filter membrane.Be sure of that salt contains about 85% R, S, R isomer.

Be to determine maximum water solubility, the dried salt of 25mg shaken in water at 37 ℃ that filtered sample is also measured UV spectrum.Observing this salt is abundant dissolved.This salt is more solvable and therefore be dissolved into 50mg/ml again in aforementioned same mode.

When solubleness when 25mg/ml dissolves is 35mM (25mg/ml)

When solubleness when 50mg/ml dissolves is 70mM (50mg/ml)

The another kind preparation of the arginic acid salt of embodiment 24-TRI50C

Arginic acid salt be by will be slightly excessive the L-arginine of mole add 0.2-0.3mmol TRI50c in the solution of 10ml ethyl acetate and easy preparation.With solvent evaporation, resistates grinds twice to remove excessive arginine with hexane after 1 hour.

The solubleness of embodiment 25-TRI50C

As above described acquisition derives from the UV/ visible spectrum and the solubleness thereof of TRI 50c of the program of embodiment 5 about salt.When the solubleness of TRI50c when 50mg/ml dissolves is 8mM (4mg/ml).

¹³C NMR 75MHz δ _C(CD ₃C(O)CD ₃)206.56，138.30，130.76，129.64，129.31，129.19，129.09，128.20，128.04，74.23，73.55，67.78，58.76，56.37，56.03，48.38，47.87，39.00，25.42，25.29。

FTIR(KBr disc)v _max(cm ^-1)3331.3，3031.4，2935.3，2876.9，2341.9，1956.1，1711.6，1639.9，1534.3，1498.1，1453.0，1255.3，1115.3，1084.6，1027.6，917.3，748.9，699.6，594.9，504.5，467.8。

Embodiment 26-(R, S, R) analysis of the sodium salt of TRI 50C and zinc salt

Following salt uses n: 1 borate: metallochemistry metering preparation, wherein n is the valency of metal, uses than (R, S, R) the TRI 50c that are used to prepare the higher chiral purity of salt among the embodiment 10.

A. sodium salt

The analytical data physical properties

HPLC or LC/MS:HPLC betabasic C18 shape: amorphous solid

Column, CH ₃CN, water

Color: white

Estimate purity:＞95%, by UV (λ _215nm) measure

Fusing point: N/A

Trace analysis:

Calculate. discovery. solubleness: solvable in aqueous medium

C： 59.24 59.93 ca～50mg/ml

H： 6.44 6.47

N： 7.67 7.31 M _w：547.40

Other: B:1.98 1.91

Na： 4.20 3.81

B. zinc salt

The analytical data physical properties

HPLC or LC/MS:HPLC betabasic C18 shape: amorphous solid

Column, CH ₃CN, water

Color: white

Estimate purity:＞95%, by UV (λ _215nm) measure

Fusing point: N/A

Trace analysis:

Calculate. discovery. solubleness: solvable in aqueous medium

C： 58.21 56.20 ca～2mg/ml

H： 6.33 6.33

N： 7.54 7.18 M _w：1114.18

Other: B:1.94 1.84

Zn： 5.87 7.26

Attention:In trace analysis, use the sour borate of trilateral chemistry formula.The sodium salt solubleness of be sure oing report in embodiment 11 is lower, because the salt of test has lower chiral purity in embodiment 11.

Conclusion:

Sodium salt and zinc salt all prepare two TRI 50c molecules a TRI 50c molecule and a metal ion with a metal ion respectively.The value of finding at sodium salt approaches and is therefore consistent with 1: 1 stoichiometry calculating.With regard to zinc salt, find excessive zinc; But zinc salt comprises the sour borate of effective ratio.

Embodiment 27-stability

Before drying and analyze TRI 50c and sodium salt and lysine salt afterwards.

Method:

Weigh TRI 50c and Na thereof, Ca and Lys salt and place the HPLC bottle were stored in moisture eliminator through dry 1 week of Vanadium Pentoxide in FLAKES.For carrying out sample analysis, the 5mg that weighs do and non-dried material places the 5mL measuring bottle, and be dissolved in the 1mL acetonitrile and water is filled to 5mL.

By HPLC research compound.At impurity profile, calculate HPLC peak area per-cent.The results are shown in table 1.

Table 1

Compound	Amount [μ g/mL]	Purity (% area)
Compound	Amount [μ g/mL]	Purity (% area)	The TRI 50c that does	1000.0	82.00
Non-dried TRI 50c	947.3	85.54	The TRI 50c that does	1000.0	82.00

The TRI 50c Na salt of doing	1024	98.81
The TRI 50c Na salt of doing	1024	98.81	Non-dried TRI 50c Na salt	1005.8	98.61
The TRI 50c Lys salt of doing	813.3	90.17	Non-dried TRI 50c Na salt	1005.8	98.61
The TRI 50c Lys salt of doing	813.3	90.17	Non-dried TRI 50c Lys salt	809.8	92.25

The purity of acid reduces by drying process, but little to the purity influence of salt; The purity of sodium salt does not significantly reduce.Yet the obvious difference in the response factors will reduce real impurity level.

This embodiment shows salt of the present invention, metal-salt an alkali metal salt for example especially, and particularly TRI 50c is more stable than acid salt.

Embodiment 28-stability

The stability of TRI 50c and TRI 50c lysine salt when this embodiment has contrasted in being filled into the casing hard gelatin capsule.

The form result

Compound	Packing	1.5 the weather condition of the moon ⁰⁾	Purity (HPLC% area) T0	Purity (HPLC% area) ³ T1
Compound	Packing	1.5 the weather condition of the moon ⁰⁾	Purity (HPLC% area) T0	Purity (HPLC% area) ³ T1	TRI50c	The foaming capsule	25℃/60％ r.h. ⁴	99	73.9
TRI50c	The foaming capsule	40℃/75％ r.h	99	73.9	TRI50c	The foaming capsule	25℃/60％ r.h. ⁴	99	73.9
TRI50c	The foaming capsule	40℃/75％ r.h	99	73.9	TRI50c	Capsule ¹	40℃/75％ r.h	99	75.3

The TRI50c lysine salt	The foaming capsule	25℃/60％ r.h.	90.2 ²⁾	90.5
The TRI50c lysine salt	The foaming capsule	25℃/60％ r.h.	90.2 ²⁾	90.5	The TRI50c lysine salt	The foaming capsule	40℃/75％ r.h	90.2 ²	91.8
The TRI50c lysine salt	Capsule ¹	40℃/75％ r.h	90.2 ²	90.6	The TRI50c lysine salt	The foaming capsule	40℃/75％ r.h	90.2 ²	91.8

Attention:

0) under specified criteria, stored 1.5 months, then with sample at room temperature storage until carrying out analytical test.

1) the storage capsule that under weather condition separately, need not bubble.

2) purity before storing batch

3) purity (capsule 's content is poured out, analyzed then) of storing batch.

4) r.h.=relative humidity

Conclusion:

Purity at T0 and T1 lysine salt does not have significant difference.

2. routine analyzer

2.1 specimen preparation

2.1.1TRI the analysis of 50c and salt

TRI 50c-standard substance (free acid) is stored in the moisture eliminator through dry 2 days of Vanadium Pentoxide in FLAKES.The reference standard of weighing then thing places measuring bottle and is dissolved in acetonitrile and the mixture of water (25/75v/v%).Gained solution equal portions (ST 1A) dilution scheme water as shown in table 4 is diluted in succession.

The stoste of Tri 50c and standardized solution

	Net weight	Purity	Salt-factor	Dissolved	Solvent	Conc.	Calibr.
	Net weight	Purity	Salt-factor	Dissolved	Solvent	Conc.	Calibr.	mg	％	Among the ml	[μg/ml]	[μg/ml]
ST 1A	40.8	98.23	1	10	ACN/ water 25/75 (v/v%)	4007.8	C4000	mg	％	Among the ml	[μg/ml]	[μg/ml]


									ml	ST	[μg/ml]	ad ml	Solvent	[μg/ml]
ST 2A	5	1A	4007，8	10	Water	2003.9	C2000		ml	ST	[μg/ml]	ad ml	Solvent	[μg/ml]
ST 2A	5	1A	4007，8	10	Water	2003.9	C2000	ST 3A	5	2A	2003，9	10	Water	1001.9	C1000
ST 4A	5	3A	1001，9	10	Water	501.0	C500	ST 3A	5	2A	2003，9	10	Water	1001.9	C1000
ST 4A	5	3A	1001，9	10	Water	501.0	C500	ST 5A	5	4A	500，9	10	Water	250.5	C250
ST 6A								ST 5A	5	4A	500，9	10	Water	250.5	C250
ST 6A		1	3A	1001，9	10	Water	100.2	C100
ST 7A		1	3A	1001，9	10	Water	100.2	C100
ST 7A		1	6A	100，2	10	Water	10.0	C10

2.1.2 the capsular impurity profile of storing

The capsule of each batch that taking-up is stored under corresponding weather condition, and the 10mg content of weighing places the 10ml measuring bottle, is dissolved in the acetonitrile/water mixture (25/75v/v%) of 10ml.Inject these solution and carry out Impurity Distribution map analysis and quantification respectively.

3. data evaluation

Use the HPLC-PDA method to carry out quantitative evaluation and Impurity Distribution map analysis.The processing wavelength set is 258nm.

4. analytical parameters

4.1 equipment and software

Self-actuated sampler Waters Alliance 2795

Pump Waters Alliance 2795

Column oven Waters Alliance 2795

Detect Waters 996 diode array, extracted wavelength 258nm

Software version Waters Millennium Release 4.0

4.2 stationary phase

Analytical column ID S71

Material X-Terra ^TMMS C ₁₈, 5 μ m

Supplier Waters, Eschbom, Germany

Yardstick 150mm * 2.1mm (length, internal diameter)

4.3 moving phase

Water: A: 0.1%HCOOH in water

Organic phase: C:ACN

Gradient condition:

Time	Flow velocity	％A	％C
Time	Flow velocity	％A	％C	0.00	0.5	90	10
27.0	0.5	10	90	0.00	0.5	90	10
27.0	0.5	10	90	27.1	0.5	90	10
30.0	0.5	90	10	27.1	0.5	90	10

Embodiment 29-TRI 50B is to the inhibition of thrombocyte procoagulant activity

When adding with the pretreated thrombocyte of zymoplasm, mixture by independent zymoplasm, independent collagen or zymoplasm and collagen is caused, in the situation that has factor Va, can observe the thrombocyte procoagulant activity increases the rate of activation of thrombogen with factor Xa.This character is because due to the increase of anionic phospholipid and the microvesicle followed discharge from the surface on the platelet surface.This is a basic physiological responses, and people's (Scott syndrome) of its thrombocyte generation procoagulant activity ability drop illustrates bleeding tendency and increases the weight of.

Method:

The washing thrombocyte 1.15nM zymoplasm, the mixture of 23 μ g/ml collagens or two kinds of materials in same concentrations 37 ℃ of processing.Add preceding 1 minute of activator or with the activator incubation after add TRI 50b immediately.Determine that as described above the thrombocyte procoagulant activity (Goodwin C A et al, Biochem J.19958,308:15-21).

TRI 50b is proved to be the powerful inhibitor of thrombocyte procoagulant activity, its IC ₅₀The following summary.

Table 2:TRI 50b is to the influence by the thrombocyte procoagulant activity of multiple agonist induction

Table 2

The IC50 that carries that agonist does not have TRI 50b adds that pre-temperature IC50 is without incubation

High multiple is educated (nM) (nM)

Zymoplasm 30 8 3000

Collagen 45 200 300

Zymoplasm/collagen 110 3 80

Table 2 for example illustrates when thrombocyte is used Thrombin treatment, and it is compared the speed that causes activating thrombogen with the contrast thrombocyte and improves 30 times.Handle at given multiple concentration level with TRI 50 and to make this acceleration reduce half.Pass through IC ₅₀The fact table of value in the nmole scope understood the remarkable effectiveness of TRI 50.

TRI 50b does not have effect to the platelet aggregation of ADP, collagen or the washing of suprarenin inductive.

The external shunting model of embodiment 30-rabbit (extracorporeal shunt model)

Introduction

This technical description a kind of animal model, wherein produced the thrombus of thrombocyte enrichment.Contrasted the activity of TRI 50b and heparin.

Use carotid artery of the rabbit of anaesthetizing and the extracorporeal circulation that jugular vein produces the external source surface (silk thread) of containing suspension.Generation by shearing force turbulent artery blood flow, platelet activation, cohesion causes thrombus and deposits under the situation that has thrombophilia (thrombogenic) surface subsequently.It is the thrombocyte enrichment that histopathological study illustrates this thrombus.

Material and method

Animal:

Use NZW rabbit (male 2.5-3.5kg).Allow animal pickuping food and water until anaesthetizing.

Anesthesia:

By give before the intramuscularly art animal premedicate altogether 0.15ml fontanel/ R-2028 (fluanisone) (Hypnorm).(10mg/ml) induces general anesthesia with methyl hexyl ketone, carries out trachea cannula subsequently.(1-2.0%) keep anaesthetizing with the isoflurane that carries in oxygen/nitrous oxide (isoflurane).

Operation is prepared:

The animal back of the body of going down on one's knees crouch is placed, and neck belly preserved skin is made arrangements for surgery.Expose left neck artery and right jugular vein.With big Portex

Conduit (yellow specification) inserts artery, cuts to appropriate length.With Silastic

Conduit inserts vein.The anastomosis comprises long " automatic analyser " line (purple/white specification) of 5cm.Use medium sized Silastic

Tube of material connects the anastomosis in arterial side.The salt solution filling was used in this anastomosis before being exposed to circulation.In the right common femoral artery intubate to measure blood pressure.

Silk thread preparation and insertion:

The silk thread that it is 3cm that identical centre contains a length.This silk thread is made up of 000 specification Gutterman suture line, makes a call to a surgeons' knot (knotting is in outside the anastomosis) endways.

Blood flow

Blood flow rate uses Doppler probe (Crystal Biotech) to determine.The silica gel probe is being disposed on carotid artery near inserting ductus arteriosus.Use temperature-sensitive paper that blood flow is recorded on the chart recorder.

The result

Table 3

Handle	Dosage	Thrombus weight after 20 minutes	Anti-thrombosis activity
Handle	Dosage	Thrombus weight after 20 minutes	Anti-thrombosis activity	Contrast	N/A	22.4±2.2mg(n＝5)
TRI 50b	10mg/kg iv	9.78±1.9mg(n＝5)	Active	Contrast	N/A	22.4±2.2mg(n＝5)
TRI 50b	10mg/kg iv	9.78±1.9mg(n＝5)	Active		3.0mg/kg iv	15.3±2.2mg(n＝5)	Active
Heparin	100u/kg iv	22.9±1.65mg(n＝4)	Non-activity		3.0mg/kg iv	15.3±2.2mg(n＝5)	Active
Heparin	100u/kg iv	22.9±1.65mg(n＝4)	Non-activity		300u/kg iv	10.5±1.4mg(n＝4)	Active (severe haemorrhage)

Discuss

Table 3 is illustrated under the artery shearing force condition, the TRI 50b that intravenously gives 3mg/kg-10mg/kg dosage significantly suppresses thrombosis and not hemorrhage, and heparin (intravenously gives the 100u/kg heparin) in the normal clinical dosage range of treatment venous thrombosis is invalid.Although the heparin of higher dosage can have effect, cause severe haemorrhage.These illustrate that TRI 50b effectively suppresses artery thrombosis and not hemorrhage result and TRI 50b to suppress the thrombocyte procoagulant activity consistent.On the contrary, the thrombin inhibitors heparin is when at about dose,equivalent (promptly suppressing artery thrombosis) when giving, produces serious hemorrhage, and this hemorrhage when using thrombin inhibitors to treat artery thrombosis, be normal.

The embodiment 31-bleeding time contrasts

The purpose of this research is the bleeding time that contrasts heparin and TRI 50b processing in proper model.It is generally accepted that heparin is a kind of inhibitor (J.Biol.Chem.1978 Oct 10 of not so good thrombocyte procoagulant activity; 253 (19): 6908-16; Miletich JP, Jackson CM, Majerus PW1:J.Clin.Invest.1983 May; 71 (5): 1383-91).

Bleeding time is determined in the hemorrhage model of rat tails give heparin and TRI 50b through intravenously after.Based on its in rat Wessler and kinetic model effectiveness and select application dose, the dosage of selection is as follows:

TRI 50b:5 and 10mg/kg

Heparin: 100 units/kg

Material and method

Anesthesia

With vetanarcol (ip. injection 30mg/ml solution, the 2.0ml/kg) anesthesia of rat with 60mg/kg.Ip. appends anesthesia if desired.

Operation is prepared

The jugular vein intubate gives test compounds.Carry out trachea cannula with suitable intubate in addition and make animal general breathing " room air ".

Compound gives

These compounds give through intravenously in suitable carriers with 1.0ml/kg dosage.Heparin gives in salt solution, and TRI 50b is dissolved in the ethanol, then gained solution is added entry and injects (1 part of ethanol is to 5 parts of water).

Technology

After giving compound 2 minutes, downcut 2mm animal afterbody tip and this afterbody is immersed in the warm saline (37 ℃) in standard " general " container, so that clear observation blood flow with new scalper.The opening entry bleeding time stops until the tail end blood flow immediately after crosscut.After stopping, observing again the afterbody blood flow 30 seconds, and hemorrhage to guarantee no longer to take place, if hemorrhagely begin once more, continue record writing time until maximum 45 minutes.

The result

Table 4 has provided hemorrhage summary as a result and has shown increase on baseline value.

Table 4

Handle	Bleeding time (minute) (± SEM ⁺)
Handle	Bleeding time (minute) (± SEM ⁺)	Salt solution	5.1±0.6
Heparin 100u/kg iv	＞40 ^*	Salt solution	5.1±0.6
Heparin 100u/kg iv	＞40 ^*	TRI 50b 5mg/kg iv	11.3±1.2
TRI50b 10mg/kg iv	30.4±5.2	TRI 50b 5mg/kg iv	11.3±1.2

^*All severe haemorrhages did not stop after 40 minutes in all animals.

The standard error of SEM=mean value

Discuss

The result illustrates TRI 50b and all is better than heparin (produce less hemorrhage) at all dosage.Should notice that when the 100u/kg heparin is compared with 5mg/kg TRI 50b the animal that heparin is handled is compared more extensive hemorrhage with the animal of accepting TRI50b; Determined before that (embodiment 25) heparin compared with the TRI 50b of 3.0mg/kg dosage at the dosage of 100u/kg, the effectiveness that suppresses artery thrombosis is relatively poor.Heparin is a kind of thrombin inhibitors at first, and relatively poor to the inhibition of thrombocyte procoagulant activity; Therefore to bring into play the result of anticoagulating active consistent by suppressing the thrombocyte blood coagulation activity except thrombin-inhibiting activity with TRI 50b for this result.

The prodrug TRI 50B of embodiment 32-TRI 50C: pharmacokinetics and absorption

Material and method

Animal

Using body weight is the rat of about 250-300g.Animal only is being used for the fasting on the same day in iv. stage.Animal ate in the last curfew of the research of carrying out oral and intraduodenal administration, and water allows to be given to when anesthesia.

Table 5:

The mouth phase

Handle	Dosage mg/kg po	Number
Handle	Dosage mg/kg po	Number	TRI 50b	20mg/kg	2
TRI 50c	20mg/kg	2	TRI 50b	20mg/kg	2

Table 6:

Phase (intraduodenal phase) in the duodenum

Handle	Dosage mg/kg po	Number
Handle	Dosage mg/kg po	Number	TRI 50b	20mg/kg	3
TRI 50c	20mg/kg	3	TRI 50b	20mg/kg	3

Dosage

Preparation (TRI 50b/TRI 50c)

Be prepared as follows preparation: the TRI 50b of 48mg/ml is dissolved in ethanol: PEG 300 (2: 3 vol: vol).Just before administration, this solution of 5 volumes and 5% kollidon (kollidon) 178F of 3 volumes are mixed.

1) two kinds of compounds is all raised with 20mg/kg dosage through port lumen or directly inject duodenum.

Compound gives in PEG/ ethanol/kollidon preparation, and said preparation is preparation before using immediately, as being described in the paragraph of " administration " at title: stoste 15.0mg/ml.This dosage is 1.33ml/kg (equaling 30mg/kg).

Method

Raise through buccal tubes

Give rat 20mg/kg.After giving about 30 minutes, rat is anaesthetized.

Give in the duodenum

After finishing anesthesia and operative procedure, compound directly is instilled in the duodenum.

Blood sampling

The mouth phase

Anaesthetizing and performing the operation the back from the 3.8%w/v trisodium citrate of carotid artery intubate blood sampling (0.81ml) to (0.09ml).Sampling for the first time is in administration after 1 hour.1.5,2,4 hours blood samplings after administration then.

Phase in the duodenum

Blood sampling: before administration, after the administration 0.25,0.5,0.75,1.0,2,3 and 4 hour.

Blood plasma

By centrifugal (3000RPM, 10 minutes) obtain and-20 ℃ of storages until analyzing.

The result

Pharmacokinetics is analyzed

Fig. 1: mouth phase clearance rate and kinetics after giving TRI 50b or its free acid (TRI 50c).

Fig. 2: mouth phase clearance rate and kinetics after in duodenum, giving TRI 50b or its free acid (TRI 50c).

Conclusion:

When giving by intraduodenal route, TRI 50b reaches the high bioavailability (peak serum concentration) of specific ionization acid.These data and the TRI50b that in blood plasma, is hydrolyzed to TRI 50c rapidly and consistent with the TRI 50c that is effective constituent.

The result of embodiment 29-32 shows that the form with salt gives TRI 50c a kind of mode for the treatment of artery thrombosis and/or venous thrombosis is provided.

Embodiment 34-human clinical research

In human clinical volunteer's research of using 2.5mg/kg iv. dosage (dosage of significant prolongation thrombin clotting time) at the most, TRI 50b (promptly uses Simplate to the Simplate bleeding time

The bleeding time of bleeding time measuring apparatus) no effect.

Recognize the borate that the invention provides as medicine from aforementioned content, be characterized in following one or more character: the amount of the oral administration biaavailability of (1) improvement; (2) Gai Liang oral administration biaavailability consistence; (3) improved stability; (4) in any case not mentioned in the prior art.

The selection of active ingredient in pharmaceutical is the work of a complexity, and it not only needs to consider biological property (comprising bioavailability), also will consider to be suitable for the physicochemical property of processing, preparing and store.Bioavailability self depends on multiple factor, generally includes body internal stability, solvation character and absorbent properties, and every factor depends on multiple physics, chemistry and/or biological behaviour conversely again potentially.

Advantageously, preferred at least product of the present invention has enough absorptions and bioavailability.With regard to commercial use, can select the lower slightly product of solubleness according to good character integrally combined.

The present invention includes following every theme:

1. the base addition salt of the acceptable boric acid of medicine shown in chemical formula (I):

Wherein:

Y comprises a kind of hydrophobicity composition, with aminoboronic acid residue-NHCH (R ⁹)-B (OH) ₂Substrate binding site to zymoplasm has affinity together; And

R ⁹By the straight chained alkyl group that one or more ehter bond interrupts, wherein the sum of oxygen and carbon atom is 3,4,5 or 6, and perhaps R ⁹Be-(CH ₂) _mW, wherein m is 2,3,4 or 5 and W be-OH or halogen (F, Cl, Br or I).

2. the salt in the 1st, wherein R ⁹It is an alkoxy-alkyl group.

3. the salt in the 1st or the 2nd, wherein YCO-comprises the amino acid of S2 sublocus bonded with zymoplasm, and this amino acid is connected at the group of N-terminal with the S3 sublocus of bind thrombin.

4. the salt in the 1st or the 2nd, wherein Y is the dipeptides of an optional N-terminal protection, the S3 of its bind thrombin and S2 binding site, the peptide bond in the acid are optional and contain in the chain or the C of nitrogen, oxygen or sulphur in the ring by optional independently ₁-C ₁₃Alkyl replaces at N-terminal, and optional by a substituting group replacement that is selected from halogen, hydroxyl and trifluoromethyl.

5. the salt in the 4th, wherein said dipeptides is the N-terminal protection.

6. the salt in the 4th or the 5th, wherein all peptide bonds in the acid all are unsubstituted.

7. each salt in the 1-6 item, wherein S3 is the R configuration in conjunction with amino-acid residue, S2 is the S configuration in conjunction with residue, fragment-NHCH (R ⁹)-B (OH) is the R configuration.

8. each salt in the 1-7 item, wherein said boric acid is about 100nM or lower to the Ki of zymoplasm.

9. the salt in the 8th, wherein said boric acid is about 20nM or lower to the Ki of zymoplasm.

10. acceptable boric acid base additive salt of the medicine shown in chemical formula (II):

Wherein:

X is that H is (to form NH ₂) or an amino protecting group;

Aa ¹Be an amino acid with alkyl side chain, this side chain contains and is no more than 20 carbon atoms and comprises the cyclic group that at least one has maximum 13 carbon atoms;

Aa ²It is the imino-acid that 4-6 ring members arranged;

R ¹Be as chemical formula-(CH ₂) _sA group the shown in-Z, wherein s is 2,3 or 4, Z is-OH-OMe ,-OEt or halogen (F, Cl, Br or I).

11. the salt in the 10th, wherein aa ¹Be selected from Phe, Dpa and hydrogenant analogue wholly or in part thereof.

12. the salt in the 10th, wherein aa ¹Be selected from Dpa, Phe, Dcha and Cha.

13. each salt in the 10-12 item, wherein aa ¹It is the R-configuration.

14. the salt in the 10th, wherein aa ¹Be (R)-Phe or (R)-Dpa.

15. the salt in the 10th, wherein aa ¹Be (R)-Phe.

16. each salt in the 10-15 item, wherein aa ²Be a residue of imino-acid shown in chemical formula (IV):

R wherein ¹¹Be-CH ₂-,-CH ₂-CH ₂-,-S-CH ₂-,-S-C (CH ₃) ₂-or-CH ₂-CH ₂-CH ₂-, when this ring was 5 or 6 members, this group was chosen wantonly at one or more-CH ₂-group is by 1-3 C ₁-C ₃Alkyl group replaces.

17. the salt in the 16th, wherein aa ²It is the S-configuration.

18. the salt in the 16th, wherein aa ²It is (S)-proline residue.

19. the 10th salt, wherein aa ¹-aa ²Be (R)-Phe-(S)-Pro.

20. each salt in the 10-19 item, wherein fragment-NH-CH (R ¹)-B (OH) ₂It is the R configuration.

21. each salt in the 10-20 item, wherein R ¹Be 2-bromotrifluoromethane, 2-chloroethyl, 2-methoxyethyl, 3-methoxycarbonyl propyl, 3-bromopropyl, 3-chloropropyl or 3-methoxycarbonyl propyl.

22. each salt in the 10-20 item, wherein R ¹It is the 3-methoxycarbonyl propyl.

23. the salt in the 10th, it is the salt of a kind of compound shown in chemical formula (VIII):

X-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH) ₂ (VIII).

24. each salt in the 10-23 item, wherein X is R ⁶-(CH ₂) _p-C (O)-, R ⁶-(CH ₂) _p-S (O) ₂-, R ⁶-(CH ₂) _p-NH-C (O)-or R ⁶-(CH ₂) _p-O-C (O)-, wherein p is 0,1,2,3,4,5 or 6, R ⁶Be a H or 5-13 member's cyclic group, optional by being selected from halogen, amino, nitro, hydroxyl, C ₅-C ₆Cyclic group, C ₁-C ₄Alkyl and the C that contains and/or be connected with cyclic group by O in the chain ₁-

C

₄1,2 or 3 substituting group of alkyl replaces, and the aforesaid alkyl group is optional by being selected from halogen, amino, nitro, hydroxyl and C ₅-C ₆A substituting group of cyclic group replaces.

25. the salt in the 24th, a wherein said 5-13 member's cyclic group is aromatic series or heteroaromatic.

26. the salt in the 25th, a wherein said 5-13 member's cyclic group are phenyl or 6 members' heteroaromatic groups.

27. each salt in the 24-26 item, wherein X is R ⁶-(CH ₂) _p-C (O)-or R ⁶-(CH ₂) _p-O-C (O)-, p is 0 or 1.

28. each salt in the 10-22 item, wherein X is a benzyloxycarbonyl.

29. each salt in the 1-28 item, it does not comprise choline or ammonium salt.

30. each salt in the 1-29 item, it comprises derived from the borate ion of boric acid and unit price counterion.

31. each salt in the 1-29 item, it is the salt of peptide boric acid and a kind of basic metal or strong basicity nitrogen-containing organic compound.

32. the 21st salt, the pKb of wherein said strong basicity organic nitrogen compound are about 7 or more, for example about 7.5 or more.

33. the 31st or 32 salt, wherein said strong basicity nitrogen-containing organic compound are guanidine, guanidine analogue or amine.

34. each salt in the 1-33 item, it is the salt of boric acid and metal.

35. each salt in the 1-28 item, it is the salt of amine shown in boric acid and a kind of basic metal, aminosugar, guanidine or the formula (XI):

Wherein n is 1-6, R ²Be H, carboxylicesters or deutero-carboxylicesters, R ³Be H, C ₁-C ₄Alkyl or natural or non-natural amino-acid residue.

36. each salt in the 1-28 item, its be boric acid and guanidine or with the salt of amine shown in the formula (IX):

37. the salt in the 36th, it is the guanidinesalt of boric acid.

38. the salt in the 37th, it is the salt of boric acid and L-arginine or L-arginine analog.

39. the salt in the 38th, wherein the L-arginine analog is the D-arginine, the perhaps D-of homoarginine or L-isomer, agmatine [(the amino butyl of 4-) guanidine], NG-nitro-L-arginine methyl ester, perhaps 2-aminopyrimidine.

40. the salt in the 37th, it is the salt of guanidine shown in boric acid and the formula (VII):

Wherein n is 1-6, R ²Be H, carboxylicesters or deutero-carboxylicesters, R ³Be H, C ₁-C ₄The residue of alkyl or natural or alpha-non-natural amino acid.

41. the 40th salt, wherein the deutero-carboxylicesters forms C ₁-C ₄Alkyl ester or acid amides.

42. the salt in the 40th or 41, compound is the L-configuration shown in its Chinese style (VII).

43. the salt in the 37th, it is the L-arginic acid salt of peptide boric acid.

44. the salt in the 36th, it is the salt of amine shown in boric acid and the formula (IX).

45. the salt in the 41st, wherein the deutero-carboxylicesters forms C ₁-C ₄Alkyl ester or acid amides.

46. the salt in the 44th or 45, amine is the L-configuration shown in its Chinese style (IX).

47. the salt in the 44th, it is the L-lysine salt of boric acid.

48. the salt of each of 1-28 item, it is a kind of sylvite.

49. each salt in the 1-28 item, it is a kind of sodium salt.

50. each salt in the 1-28 item, it is a kind of lithium salts.

51. each salt in the 1-28 item, it is a kind of aminosugar salt.

52. the salt in the 51st, wherein said aminosugar are the sugar of open loop.

53. the salt in the 52nd, wherein said aminosugar is a glycosamine.

54. the salt in the 51st, wherein said aminosugar are a kind of annular aminosugars.

55. the salt in the 51-54 item, wherein said aminosugar are that N-terminal is unsubstituted.

56. each salt in the 51-54 item, wherein said aminosugar is replaced at N-terminal by one or two substituting group.

57. the salt in the 56th, wherein said substituting group or each substituting group all are hydrocarbyl groups.

58. the salt in the 57th, wherein said substituting group or each substituting group all are to be selected from C ₁, C ₂, C ₃, C ₄, C ₅, C ₆, C ₇And C ₈A group of alkyl group.

59. each salt in the 56-58 item wherein has a single N-terminal to replace.

60. the salt in the 51st, wherein said glycosamine are N-methyl D-glycosamines.

61. each salt in the 1-60 item, it comprises the borate ion derived from peptide boric acid, and has the stoichiometry consistent with the borate ion that carries single negative charge.

62. each salt in the 1-60 item, wherein said salt is made up of acid salt substantially, and wherein the borate group B-OH group keeps protonated when representing with trilateral.

63. each salt in the 1-62 item, wherein said salt comprise a borate ion and a counterion derived from peptide boric acid, wherein said salt is made up of the salt of the counterion with single type substantially.

64. each salt in the 1-28 item, it is a kind of single lithium salts or single sodium salt.

65. the salt in the 23rd, it is a kind of single lithium salts or single sodium salt.

66. the Cbz-of a solid phase (R)-Phe-(S)-Pro-(R)-Mpg-B (OH) ₂Single sodium salt or single lithium salts, it contains weight and is no more than 1% water.

67. the salt in the 66th, it contains weight and is no more than 0.5% water.

68. the salt in the 66th, it contains weight and is no more than 0.1% water.

69. each salt in the 66-68 item, it is single sodium salt.

70. each salt in the 1-65 item, it is a solid phase.

71. the salt in the 70th, it is a substantially dry.

72. the product as medicine, it comprises in the 1-71 item each salt.

73. the pharmaceutical preparation of an oral dosage form, it comprises in the 1-71 item each salt and a kind of medicine acceptable diluent, vehicle or carrier.

74. the preparation in the 73rd, it is a kind of solid preparation.

75. the pharmaceutical preparation in the 74th, it is suitable for discharging described salt in duodenum.

76. the pharmaceutical preparation in the 75th, it is the casing preparation.

77. the application of each salt in the thrombotic medicine of production for treating in the 1-71 item.

78. the application in the 77th, wherein said disease are a kind of acute coronary syndromes.

79. the application in the 77th, wherein said disease is Acute Myocardial Infarction.

80. the application in the 77th, wherein said disease is a venous thromboembolism, is selected from dvt and forms and pulmonary infarction.

81. the application of each salt in producing oral pharmaceutical in the 1-71 item, described oral pharmaceutical are accepted patient's medium sized vein thromboembolism of chemotherapy in order to the thrombosis in the prevention blood samples of patients dialyzing access, the cardiovascular disorder among the prevention end-stage renal disease patient, prevention by inlying catheter or are prevented through the thromboembolism among the patient of artery of lower extremity reconstruction operations.

82. each salt is being produced in order to the application in the oral pharmaceutical of treatment or prevention of arterial disease in the 1-71 item, described artery disease is selected from acute coronary syndrome, cerebrovascular thrombosis, peripheral arterial obturation and derives from the artery thrombosis of atrial fibrillation, valvular heart disease, arteriovenous branch road, inlying catheter or coronary stricture.

83. an oral drug preparation, it comprises in (i) 1-71 item each salt, and (ii) a kind of combination of pharmaceutically active agents.

84. an oral drug preparation, it comprises in (i) 1-71 item each salt, and the combination of (ii) another kind of cardiovascular treatment agent.

85. the preparation in the 84th, wherein another kind of cardiovascular treatment agent comprise fat-reducing medicament, the special class medicine of shellfish, nicotinic acid, statin, CETP inhibitor, cholic acid chelating agent, antioxidant, IIb/IIIa antagonist, aldosterone inhibitor, A2 antagonist, A3 agonist, beta blocker, acetylsalicylic acid, loop diuretic, ace inhibitor, the antithrombotic agent with different mechanism of action, anti-platelet agent, thromboxane acceptor and/or synthetase inhibitors, fibrinogen deceptor antagonists, prostacyclin mimetics, phosphodiesterase inhibitor, adp receptor (P ₂T) antagonist, thrombus dissolving preparation, cardioprotective agent or cox 2 inhibitor.

86. each preparation in the 83-85 item, it is a substantially dry.

87. the application of each salt in producing a kind of medicine for example gives described medicine and another kind of cardiovascular treatment agent jointly with preventing cardiovascular disease in the 1-71 item.

88. oral pharmaceutical, it comprises a kind of borate, described boric acid is a kind of selective thrombin inhibitors and the hydrophobic group that has a neutral aminoboronic acid residue and be connected with this neutrality aminoboronic acid residue through a peptide bond, described neutral aminoboronic acid residue energy bind thrombin S1 sublocus, described hydrophobic group energy bind thrombin S2 and S3 sublocus, described salt comprises the positively charged ion of a n valency, and it observes stoichiometry and theoretical chemistry metering n: 1 (boric acid: positively charged ion) consistent.

89. the medicine in the 88th, wherein said boric acid is about 100nM or lower to the Ki of zymoplasm, optional about 20nM or lower.

90. one kind is suitable for oral pharmaceutical preparation, it comprises a kind of compound of solid phase, and this compound is corresponding to sour Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B (OH) ₂Borate substance the source and be the acceptable cationic source except choline and ammonium of medicine.

91. one kind forms the boric acid shown in the formula (I) or can be the method for the product in source in this boric acid body:

Wherein:

R ⁹Be a straight chained alkyl group that is interrupted by one or more ehter bond, wherein the sum of oxygen and carbon atom is 3,4,5 or 6, and perhaps R ⁹Be-(CH ₂) _mW, wherein m is 2,3,4 or 5, W is-OH or halogen (F, Cl, Br or I);

Described method comprises:

The diethyl ether solution of the ester of described acid is provided;

Diethanolamine is dissolved in this solution;

Allow or cause a kind of precipitation to form and reclaim this precipitation; And

Change throw out into a kind of end product, described end product is selected from free organic boronic or can is a kind of product of originating in the body of this boric acid.

92. the method in the 91st, it further comprises described end product is formulated as pharmaceutical composition.

93. a method that forms each salt in the 1-71 item comprises that acetonitrile solution and the acceptable alkali of a kind of medicine with corresponding boric acid makes up to form salt.

94. one kind makes the stable method of a kind of organic boronic, comprises with its salt form providing.

95. one kind the organic boronic medicine presented method as medicinal application with stable form, comprises the organic boronic medicine of acceptable its base addition salt form of the medicine that solid phase is provided.

96. be suitable for oral a kind of pharmaceutical preparation, it comprises:

A) be selected from first material of the equilibrium form of the borate ion of boric acid, described boric acid shown in the formula (I) and described boric acid and described borate ion:

Wherein:

R ⁹Be a straight chained alkyl group that is interrupted by one or more ehter bond, wherein the sum of oxygen and carbon atom is 3,4,5 or 6, and perhaps R ⁹Be-(CH ₂) _mW, wherein m is 2,3,4 or 5, W is-OH or halogen; And

(b) be selected from second material of the acceptable metal ion of medicine, Methionin, arginine and aminosugar, the valency of described metal ion is n;

Wherein when described second kind of material be valency when being 1 metal ion or Methionin, arginine or aminosugar, first kind of observation stoichiometry and 1: 1 basically identical of theoretical chemistry metering with second kind of material in the described preparation; When described second kind of material is valency when being higher than 1 metal ion, the observation stoichiometry is n: 1.

Claims

1. the acceptable base addition salt of medicine of a boric acid and metal or strong basicity nitrogen-containing organic compound, wherein said boric acid are suc as formula (II):

Wherein:

X is H or amino protecting group;

Aa ¹Be a seed amino acid, it has and contains an alkyl side chain that is no more than 20 carbon atoms and comprise the cyclic group that at least one has maximum 13 carbon atoms;

Aa ²Be have a kind of imino-acid of 4-6 ring members or by one by C ₃-C ₁₃The Gly that hydrocarbyl group N-replaces;

R ¹Be as chemical formula-(CH ₂) _sA group the shown in-Z, wherein s is 2,3 or 4, Z is-OH ,-OMe ,-OEt or halogen F, Cl, Br or I.

2. the salt of claim 1, wherein aa ¹Be selected from Phe, Dpa and all or part of hydrogenant analogue thereof.

3. the salt of claim 1, wherein aa ¹Be selected from Dpa, Phe, Dcha and Cha.

4. the salt of claim 1, wherein aa ¹It is (R)-configuration.

5. the salt of claim 1, wherein aa ¹Be (R)-Phe or (R)-Dpa.

6. the salt of claim 1, wherein aa ¹Be (R)-Phe.

7. the salt of claim 1, wherein aa ²Be a kind of imino-acid residue shown in the chemical formula (IV):

R wherein ¹¹Be-CH ₂-,-CH ₂-CH ₂-,-S-CH ₂-,-S-C (CH ₃) ₂-or-CH ₂-CH ₂-CH ₂-, when described ring is 5 or 6 yuan of whens ring, this imino-acid one or more-CH ₂-group place is optional by 1-3 C ₁-C ₃Alkyl group replaces.

8. the salt of claim 1, wherein aa ²It is (S)-configuration.

9. the salt of claim 7, wherein aa ²It is (S)-proline residue.

10. the salt of claim 1, wherein aa ¹-aa ²Be (R)-Phe-(S)-Pro.

11. the salt of claim 1, wherein residue-NH-CH (R ¹)-B (OH) ₂It is the R configuration.

12. the salt of claim 1, wherein aa ¹Be (R)-configuration, and residue-NH-CH (R ¹)-B (OH) ₂It is (R)-configuration.

13. the salt of claim 12, wherein aa ¹Be selected from Phe, Dpa and all or part of hydrogenant analogue thereof, aa ²Be a kind of imino-acid residue shown in the chemical formula (IV):

R wherein ¹¹Be-CH ₂-,-CH ₂-CH ₂-,-S-CH ₂-,-S-C (CH ₃) ₂-or-CH ₂-CH ₂-CH ₂-, when described ring is 5 or 6 yuan of whens ring, this imino-acid one or more-CH ₂-group place is optional by 1-3 C ₁-C ₃Alkyl group replaces, and R ¹It is the 3-methoxy-propyl.

14. the salt of claim 12, wherein R ¹Be 2-bromotrifluoromethane, 2-chloroethyl, 2-methoxy ethyl, 3-bromopropyl, 3-chloropropyl or 3-methoxy-propyl.

15. the salt of claim 12, wherein R ¹It is the 3-methoxy-propyl.

16. the salt of claim 1, it is the salt of the compound of formula (VIII):

X-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH) ₂ (VIII)。

17. the salt of claim 11, wherein X is R ⁶-(CH ₂) _p-C (O)-, R ⁶-(CH ₂) _p-S (O) ₂-, R ⁶-(CH ₂) _p-NH-C (O)-or R ⁶-(CH ₂) _p-O-C (O)-, wherein p is 0,1,2,3,4,5 or 6, R ⁶Be the cyclic group of H or 5-13 unit, this cyclic group is randomly replaced by 1,2 or 3 substituting group, and described substituting group is selected from halogen, amino, nitro, hydroxyl, C ₅-C ₆Cyclic group, C ₁-C ₄Alkyl and the C that contains the interior O of chain and/or be connected with described cyclic group by O in the chain ₁-C ₄Alkyl, the aforesaid alkyl group is optional by being selected from halogen, amino, nitro, hydroxyl and C ₅-C ₆The substituting group of cyclic group replaces.

18. the salt of claim 17, the first cyclic group of wherein said 5-13 is aromatic ring or hetero-aromatic ring.

19. the salt of claim 18, the first cyclic group of wherein said 5-13 is phenyl or 6 yuan of hetero-aromatic ring groups.

20. the salt of claim 12, wherein X is R ⁶-(CH ₂) _p-C (O)-, R wherein ⁶Defined in claim 17, and p is 0 or 1.

21. the salt of claim 1, wherein X is a benzyloxycarbonyl.

22. the salt of claim 15, wherein X is a benzyloxycarbonyl.

23. the salt of claim 1, its mesoboric acid are Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B (OH) ₂

24. the salt of claim 12, it does not comprise choline or ammonium salt.

25. the salt of claim 12, it is made up of borate ion and unit price counterion derived from boric acid.

26. the salt of claim 1, the pKb of wherein said strong basicity organic nitrogen compound are 7 or higher.

27. the salt of claim 12, it is a borate metal salt.

28. the salt of claim 1, it is the salt of the amine shown in boric acid and basic metal, aminosugar, guanidine or the chemical formula (XI):

Wherein n is 1-6, R ²Be H, carboxylate radical, R ³Be H, C ₁-C ₄The residue of alkyl or a kind of natural or alpha-non-natural amino acid.

29. the salt of claim 23, it is the L-arginic acid salt.

30. the salt of claim 23, it is the L-lysine salt.

31. the salt of claim 23, it is a sylvite.

32. the salt of claim 23, it is a sodium salt.

33. the salt of claim 23, it is a lithium salts.

34. the salt of claim 23, it is a glucosamine salt.

35. the salt of claim 34, wherein said glycosamine are N-methyl D-glycosamines.

36. the salt of claim 12, it comprises the borate ion derived from peptide boric acid, and has the stoichiometry consistent with the borate ion that carries a single negative charge.

37. the salt of claim 12, wherein said salt is become with the balance ionic group by the borate ion derived from peptide boric acid, and wherein said salt is made up of the salt of the counterion with single type substantially.

38. the salt of claim 13, it is single lithium salts, single sodium salt or monopotassium salt.

39. the salt of claim 13, it is lithium salts or sodium salt.

40. the salt of claim 13, it is single sodium salt.

41.Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B (OH) ₂An alkali metal salt.

42.Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B (OH) ₂Single an alkali metal salt.

43.Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B (OH) ₂Single sodium salt.

44. the salt of claim 1, it is a solid phase.

45. the salt of claim 32, it is a solid phase.

46. the salt of claim 41, it is a solid phase.

47. the salt of claim 1, it is in solution.

48. the salt of claim 13, it is an an alkali metal salt and in solution.

49. the salt of claim 32, it is in solution.

50. the salt of claim 12, it comprises the borate of acid anhydride form.

51. as a kind of product of medicine, it comprises each salt of claim 1-50.

52. each the aqueous solution of salt of claim 1-43.

53. the pharmaceutical preparation of oral dosage form, it comprises each salt of claim 1-50.

54. the preparation of claim 53, it further comprises medicine acceptable diluent, vehicle or carrier.

55. be suitable for oral a kind of pharmaceutical preparation, it comprises:

A) be selected from (a) as the borate anion of boric acid as described in the boric acid of each formula that is limited (I) of claim 1-20, (b) and (c) boric acid and as described in first kind of material of equilibrium form of borate anion: and

B) being selected from metal ion and pKb is 7 or second kind of acceptable material of medicine of higher alkaline nitrogen-containing organic compound.

56. the preparation of claim 55, wherein said first kind of material is selected from acid anhydride class material.

57. the preparation of claim 55, wherein said second kind of material is selected from alkalimetal ion.

58. the preparation of claim 55, wherein said second kind of material is selected from L-arginine, L-Methionin or N-methyl D-glycosamine positively charged ion.

59. the preparation of claim 55, wherein said second kind of material is selected from sodium ion.

60. the preparation of claim 55, it is made up of first kind of material, second kind of material and the acceptable member of medicine and optional another kind of cardiovascular treatment agent basically, and the acceptable member of described medicine is selected from the material of thinner, vehicle, carrier, casing and formation packing.

61. claim 1-42 each salt or each preparation of claim 53-60 in preparation by prevention or treat application in the thrombotic medicine.

62. the application of claim 61, wherein said medicine is used for the treatment of the venous thromboembolism incident.

63. claim 1-50 each salt or each preparation of claim 53-60 in the application of preparation in a kind of oral pharmaceutical, described medicine is used for preventing the thrombosis of blood samples of patients dialyzing access, be used to prevent end-stage renal disease patient's thrombus cardiovascular event, be used to prevent to accept the patient's vein thromboembolic events of chemotherapy by inlying catheter, perhaps be used for preventing artery of lower extremity reconstructive procedure patient thromboembolic events, or pass through treatment or prevent to treat artery disease, described artery disease is selected from acute coronary syndrome, cerebrovascular thrombosis, peripheral arterial obturation and generation are from atrial fibrillation, the valve heart disease, arteriovenous shut, the artery thrombosis of inlying catheter or coronary artery bracket.

64. each salt or the application that is used for when blood contacts with medical apparatus outside the body, carrying out the medicine that anticoagulation handles in preparation of each preparation of claim 53-60 of claim 1-50.

65. oral drug preparation, its comprise (i) claim 1-50 each salt and the (ii) combination of another kind of pharmaceutically active agents, wherein said another kind of pharmaceutically active agents is a kind of cardiovascular treatment agent, and it is selected from by lipid lowerers, the special class medicine of shellfish, nicotinic acid, statin, the CETP inhibitor, cholic acid chelating agent, antioxidant, the IIb/IIIa antagonist, the aldosterone inhibitor, the A2 antagonist, the A3 agonist, beta blocker, acetyl salicylic acid, loop diuretic, the ace inhibitor, antithrombotic agent with different mechanism of action, anti-platelet agents, thromboxane acceptor and/or synthetase inhibitors, fibrinogen deceptor antagonists, prostacyclin mimetics, phosphodiesterase inhibitor, ADP-acceptor (P ₂T) in the group that antagonist, thrombolytics, cardioprotective agent and cox 2 inhibitor are formed.

66. a pharmaceutical preparation that is suitable for orally give, it comprises a kind of solid-phase compound, and this compound is corresponding to sour Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B (OH) ₂The borate material the source and be the acceptable cationic source of medicine except choline and ammonium.

67. the preparation of claim 66, wherein said compound is selected from the acid anhydride class.

68. one kind forms each the method for salt of claim 1-50, comprising:

Diethanolamine is dissolved in a kind of ethereal solution of diol ester of described acid;

Make or impel a kind of precipitation to form and reclaim this precipitation;

This throw out is converted into free organic boronic; And

This organic boronic and the acceptable alkali reaction of a kind of medicine are formed described salt.

69. the method for claim 68, wherein said ether are that diethyl ether and described ester are pinacol esters.

70. a method for preparing oral drug preparation comprises:

With each defined boric acid of claim 1-20 and pKb be 7 or the higher acceptable alkali of medicine contact; And

Products therefrom is mixed with preparation.

71. the method for claim 70, it further comprises and obtains organic boronic as follows:

Form the solution of the diol ester of each defined boric acid of claim 1-20;

Make up described solution and diethanolamine, and make or impel described diethanolamine and the insoluble precipitation of described ester reaction formation;

Reclaim described precipitation;

Described precipitation is converted into free boric acid.

72. the method for claim 71, the solvent of wherein said solution is an ether.

73. the method for claim 72, wherein said ester is a pinacol ester.

74. the method for claim 70, wherein said alkali are pKb is 7 or higher nitrogen-containing organic compound, or metal base.

75. the method for claim 74, wherein said alkali pKb is 7.5 or is more greatly and not choline or ammonia.

76. the method for claim 70, wherein said alkali is alkali metal base.

77. the method for claim 76, wherein said alkali is soda.

78. the method for claim 74, wherein said alkali are L-arginine, L-Methionin or N-methyl D-glycosamine.

79. the method for claim 76, wherein said boric acid are Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B (OH) ₂

80. the method for claim 70, wherein said product comprises the boron trioxide class.

81. the application of the diol ester of each defined boric acid of claim 1-20 in being prepared as follows oral drug preparation, the boric acid that the described hydrolysis that is prepared as the diethanolamine ester that the transesterificationization that makes by glycol ester hydrolysis or described diol ester makes produces be the acceptable alkali reaction of a kind of medicine of the amine shown in alkali metal base, aminosugar, guanidine or the chemical formula (XI) and products therefrom be formulated as pharmaceutical preparation:

82. the application of claim 81, the wherein said ester that is hydrolyzed are pinacol ester, pinane diol ester or diethanolamine ester.

83. the application of claim 81, wherein said alkali are alkali metal base, L-arginine, L-Methionin or N-methyl D-glycosamine.